Fibrotic treatment

ABSTRACT

The present invention relates to a method for the treatment of fibrosis, in particular cardiac fibrosis, comprising the administration of an inhibitor of insulin-regulated aminopeptidase (IRAP). Preferable the IRAP inhibitor is chosen from the group including HFI-419, HA-08, AL-40, HFI-437, Val-Tyr-Ile-His-Pro-Phe (otherwise known as angiotensin IV or ANG IV), c[Cys-Tyr-Cys]-His-Pro-Phe, and c[Hcy-Tyr-Hcy]-His-Pro-Phe.

CROSS REFERENCE(S) TO RELATED APPLICATIONS

This application claims priority from Australian provisional applicationno. 2015903035, the entire contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to compositions, methods and kits for thetreatment of fibrosis. In particular, the compositions, methods and kitsare particularly useful, but not limited to, the treatment of organfibrosis.

BACKGROUND OF THE INVENTION

Cardiovascular diseases (CVDs) remain the world's leading cause ofmorbidity and mortality, claiming 17 million deaths annually, accountingfor 1 death every 2 s worldwide. Importantly, prevalence of major CVDsincreases exponentially after the age of 60, with aged patients oftensuffering from cardiac dysfunction or chronic heart failure (CHF). CVDsare often initiated upon any cardiac insult or injury, which thentriggers the innate defense mechanism and inflammatory response tocounter-regulate and repair the injury, in a process known as cardiacremodeling. However, repetitive injury or dysregulated reactiveremodeling eventually leads to accumulation of excessive collagens inthe heart, driving towards a progressively irreversible fibroticresponse, leading to permanent scarring or cardiac fibrosis.Subsequently, blood supply to the heart is impaired, while increasedstiffness of the heart further hinders cardiac contractility whichpredisposes to myocardial infarction (MI), chronic heart failure (CHF)or end organ damage. Such events are more likely to occur in the agingpopulation, thus further increasing the susceptibility towardsmyocardial infarction or injury, with ageing itself compromised by theinefficient reparative process. Moreover, there are few treatmentsavailable which are directed against fibrosis. Of these, angiotensinconverting enzyme (ACE) inhibitor or angiotensin receptor blockers(ARBs) only reduced CV mortality rate by 7%.

Fibrosis can occur in various tissues, such as the heart (as discussedabove), lungs, liver, skin, blood vessels and kidneys.

There is a need for therapies for the treatment and/or prevention offibrosis.

Reference to any prior art in the specification is not an acknowledgmentor suggestion that this prior art forms part of the common generalknowledge in any jurisdiction or that this prior art could reasonably beexpected to be understood, regarded as relevant, and/or combined withother pieces of prior art by a skilled person in the art.

SUMMARY OF THE INVENTION

The present invention provides a method of treating fibrosis in anindividual comprising administering an inhibitor of insulin-regulatedaminopeptidase (IRAP), thereby treating fibrosis. Preferably, theindividual is identified as having fibrosis.

In any aspect of the present invention, the method or use reducesprogression of at least one clinically or biochemically observablecharacteristic of fibrosis, thereby treating fibrosis.

In any aspect of the present invention, the method or use reverses atleast one clinically or biochemically observable characteristic offibrosis, thereby treating fibrosis.

The clinically or biochemically observable characteristic may be any oneor more of the following organ dysfunction, scarring, alteration ofnormal extracellular matrix balance, increase in collagen deposition,differentiation of fibroblasts to myofibroblasts, reduction in the levelof matrix metalloproteinases and increase in the level of tissueInhibitors of matrix metalloproteinases. Preferably, collagen is aprecursor or mature forms of collagen α1 Type 1.

In any aspect of the invention, the fibrosis may be age-induced,injury-induced or stress-induced. Preferably, the fibrosis is selectedfrom the group consisting of cardiac fibrosis, liver fibrosis, kidneyfibrosis, vascular fibrosis, lung fibrosis and dermal fibrosis.

In any method of the invention, the method further comprises the step ofidentifying an individual having fibrosis.

In any aspect of the invention, the inhibitor of IRAP inhibits IRAPmediated signalling. Typically, the inhibitor of IRAP directly inhibitsthe enzymatic activity of IRAP. Preferably, the inhibitor binds to theactive site of IRAP. More preferably, the inhibitor of IRAP competeswith, or prevents the binding of a substrate of IRAP for binding toIRAP.

The inhibitor of IRAP may exhibit a Ki value of less than 1 mM,preferably less than 100 μM, more preferably less than 101 μM, asdetermined by an assay as described herein, for example an assay thatdetermines aminopeptidase activity or substrate degradation.

Preferably the assay involves human IRAP. Typically, the assay of aminopeptidase activity comprises hydrolysis of the synthetic substrateL-Leucine 7-amido-4-methyl coumarin hydrochloride (Leu-MCA) monitored byrelease of the fluorogenic product MCA by IRAP, preferably human IRAP.The assay of substrate degradation may be degradation of the peptidesubstrates CYFQNCPRG (SEQ ID NO: 1) or YGGFL (SEQ ID NO: 2).

An inhibitor of IRAP may be selected from the group consisting of asmall molecule, an antibody, a peptide or an interfering RNA.

The invention also provides a method of alleviating or ameliorating asymptom of fibrosis in a subject in need thereof, the method comprisingadministering to the subject in need thereof a therapeutically effectiveamount of an inhibitor of IRAP, thereby alleviating or ameliorating asymptom of fibrosis in the subject. Preferably, the fibrosis isage-induced, as a result of underlying tissue injury or cardiovasculardisease.

The invention also provides use of an inhibitor of IRAP in themanufacture of a medicament for the treatment or prevention of fibrosisin a subject in need thereof.

The present invention provides a method for the treatment of fibrosis ina subject comprising the steps of

identifying a subject having fibrosis; and

administering to the subject in need thereof a therapeutically effectiveamount of an inhibitor of IRAP,

thereby treating fibrosis in the subject.

The invention has particular application to a subject having organdysfunction, scarring, alteration of normal extracellular matrixbalance, increase in collagen deposition, increased collagen volumefraction, differentiation of fibroblasts to myofibroblasts, reduction inthe level of matrix metalloproteinases and increase in the level oftissue Inhibitors of matrix metalloproteinases, increased levels ofeither N-terminal or C-terminal propeptide of type I procollagen (PINPor PICP), decreased levels of C-terminal telepeptide of Type I collagen(CTP or CITP), increased collagen deposition and impaired cardiacfunction measured by various non-invasive imagining techniques, andimpaired renal function as measured by increased proteinurea andalbuminurea, decreased glomerular filtration rate or doubling ofcreatinine levels.

The present invention provides a method for the treatment of age-inducedfibrosis or organ fibrosis related to tissue injury, the methodcomprising the steps of

identifying a subject having age-induced fibrosis or organ fibrosisrelated to tissue injury; and

administering to the subject in need thereof a therapeutically effectiveamount of an inhibitor of IRAP,

thereby treating age-induced fibrosis or organ fibrosis related totissue injury.

In any aspect or embodiment of the invention, age-induced fibrosis maybe reference to age-induced fibrosis of the heart (cardiac), kidney(renal), blood vessels (vascular), liver (hepatic), pancreas and lung(pulmonary).

The present invention provides a method for the treatment or preventionof fibrosis, the method comprising the step of administering acomposition to the subject for treatment or prevention, wherein thecomposition comprises, consists essentially of or consists of aninhibitor of IRAP and a pharmaceutically acceptable diluent, excipientor carrier.

In any method or use of the invention described herein, an inhibitor ofIRAP may be administered systemically or directly to the site ofdisease. The inhibitor of IRAP may be formulated for oraladministration.

The invention provides a pharmaceutical composition for treating orpreventing fibrosis comprising an inhibitor of IRAP and apharmaceutically acceptable diluent, excipient or carrier. In oneembodiment, the only active ingredient present in the composition is aninhibitor of IRAP.

The invention provides a pharmaceutical composition for treating orpreventing fibrosis comprising as an active ingredient an inhibitor ofIRAP and a pharmaceutically acceptable diluent, excipient or carrier. Inone embodiment, the only active ingredient present in the composition isan inhibitor of IRAP.

The invention provides a pharmaceutical composition for treating orpreventing fibrosis comprising as a main ingredient an inhibitor of IRAPand a pharmaceutically acceptable diluent, excipient or carrier. In oneembodiment, the only active ingredient present in the composition is aninhibitor of IRAP.

The invention also provides an inhibitor of IRAP for use in thetreatment of fibrosis.

The invention also provides a pharmaceutical composition comprising aninhibitor of IRAP and a pharmaceutically acceptable diluent, excipientor carrier for use in the treatment of fibrosis.

In one aspect of the present invention, the inhibitor of IRAP has astructure according to Formula (I):

wherein

-   -   A is aryl, heteroaryl carbocyclyl or heterocyclyl, each of which        may be optionally substituted, when R¹ is NHCOR₈;        -   or quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl,            quinoxalinyl, 1,8-naphthyridyl, phthalazinyl or pteridinyl,            each of which may be optionally substituted, when R¹ is            NR₇R₈, NHCOR₈, N(COR₈)₂, N(COR₇)(COR₈), N═CHOR₈ or N═CHR₈;    -   X is O, NR′ or S, wherein R′ is hydrogen, optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        alkynyl, optionally substituted aryl, optionally substituted        acyl, optionally substituted heteroaryl, optionally substituted        carbocyclyl or optionally substituted heterocyclyl;    -   R₇ and R₈ are independently selected from hydrogen, optionally        substituted alkyl, optionally substituted aryl, or R₇ and R₈,        together with the nitrogen atom to which they are attached form        a 3-8-membered ring which may be optionally substituted;    -   R² is CN, CO₂R⁹, C(O)O(O)R⁹, C(O)R⁹ or C(O)NR⁹R¹⁰ wherein R⁹ and        R¹⁰ are independently selected from alkyl, alkenyl, alkynyl,        aryl, heteroaryl, carbocyclyl, heterocyclyl, each of which may        be optionally substituted, and hydrogen; or R⁹ and R¹⁰ together        with the nitrogen atom to which they are attached, form a        3-8-membered ring which may be optionally substituted;    -   R₃-R₆ are independently selected from hydrogen, halo, nitro,        cyano alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl,        carbocyclyl, hydroxy, alkoxy, alkenyloxy, alkynyloxy,        alkynyloxy, aryloxy, heteroaryloxy, heterocyclyloxy, amino,        acyl, acyloxy, carboxy, carboxyester, methylenedioxy, amido,        thio, alkylthio, alkenylthio, alkynylthio, arylthio,        heteroarylthio, heterocyclylthio, carbocyclylthio, acylthio and        azido, each of which may be optionally substituted where        appropriate, or any two adjacent R³-R⁶, together with the atoms        to which they are attached, form a 3-8-membered ring which may        be optionally substituted; and    -   Y is hydrogen or C₁₋₁₀alkyl,

or a pharmaceutically acceptable salt or solvate thereof.

In any aspect of the present invention, the inhibitor of IRAP has astructure according to Formula (II):

wherein

-   -   A is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl,        heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each        of which may be optionally substituted;    -   R_(A) and R_(B) are independently selected from hydrogen, alkyl        and acyl;    -   R₁ is selected from CN or CO₂R_(C);    -   R₂ is selected from CO₂R_(C) and acyl;    -   R₃ is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl,        heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each        of which may be optionally substituted; or    -   R₂ and R₃ together form a 5-6-membered saturated        keto-carbocyclic ring:

-   -   -   wherein n is 1 or 2;        -   and which ring may be optionally substituted one or more            times by C₁₋₆alkyl; or

    -   R₂ and R₃ together form a 5-membered lactone ring (a) or a        6-membered lactone ring (b)

-   -   -   wherein            is an optional double bond and R′ is alkyl.

    -   R_(C) is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl,        heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each        of which may be optionally substituted;

or a pharmaceutically acceptable salt, solvate or prodrug thereof.

In any aspect of the present invention, the inhibitor of IRAP has astructure according to Formula (III):

wherein

R₁ is H or CH₂COOH; and

n is 0 or 1; and

m is 1 or 2; and

W is CH or N;

or a pharmaceutically acceptable salt, solvate or prodrug thereof.

In one embodiment, the inhibitor has the structure:

In another embodiment of the present invention, the inhibitor of IRAPhas a structure according to any one of the following sequences:

(SEQ ID NO: 3) Val-Tyr-Ile-His-Pro-Phe, (SEQ ID NO: 4)c[Cys-Tyr-Cys]-His-Pro-Phe, and (SEQ ID NO: 5)c[Hcy-Tyr-Hcy]-His-Pro-Phe.

In yet another embodiment of the present invention, the inhibitor has astructure according to the compound

In any aspect of the present invention, the inhibitor of IRAP may be anycompound or inhibitor as described herein.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

Further aspects of the present invention and further embodiments of theaspects described in the preceding paragraphs will become apparent fromthe following description, given by way of example and with reference tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: IRAP deficiency and IRAP inhibition attenuate AngiotensinII-induced increase in systolic blood pressure (SBP). Mean data ofsystolic blood pressure of adult WT and IRAP^(−/−) mice treated withsaline or Ang II (800 ng/kg/min)±vehicle/HFI 419 (n=6-9). Data expressedas mean±s.e.m; **P<0.01, ***P<0.001, ****P<0.0001 determined by two wayrepeated measures analysis of variance (ANOVA).

FIG. 2: IRAP expression is increased in aortae and hearts of AngiotensinII-infused WT mice. (a) Quantification of IRAP expression in medial andadventitial regions of 5 μm thick transverse aortic sections from adult(4-6 month old) WT and IRAP^(−/−) mice treated with AngII±vehicle/HFI-419 (n=5). (b) Quantification of IRAP in 5 μm thicktransverse heart sections from adult (4-6 month old) WT and IRAP^(−/−)mice treated with Ang II±vehicle/HFI-419 (n=5). Quantification of IRAPexpressed as percent positive stained tissue area. Data expressed asmean±s.e.m; **P<0.01, ***P<0.001, ****P<0.0001 determined by two wayanalysis of variance (ANOVA).

FIG. 3: Genetic deletion and pharmacological inhibition of IRAPattenuates Angiotensin II-mediated aortic fibrosis and associatedmarkers. Representative images and quantification of positive stainedimmunofluorescence in thoracic aortic sections from adult (4-6 monthold) WT and IRAP^(−/−) mice treated with saline or AngII±vehicle/HFI-419 showing decreased TGF-β₁ and α-SMA expression in redwith green showing autofluorescence of elastic lamina. Collagen stainingwas determined using picrosirius red stain and then imaged usingpolarised microscopy. Data expressed as mean±s.e.m of percentagepositive stained area (n=5-6). *P<0.05; **P<0.01; ***P<0.001,****P<0.0001 determined by one way ANOVA with Bonferroni correction formultiple comparisons.

FIG. 4: Genetic deletion and pharmacological inhibition of IRAPattenuates Angiotensin II-mediated inflammation in the aorta.Representative images and quantification of positive stainedimmunofluorescence in thoracic aortic sections from adult (4-6 monthold) WT and IRAP^(−/−) mice treated with saline or AngII±vehicle/HFI-419 showing P-IκBα (marker for NFκB activation), MCP-1,ICAM-1 and VCAM-1 (vascular cell adhesion protein-1) expression in redwith green showing autofluorescence of elastic lamina. Data expressed asmean±s.e.m of percentage positive stained area (n=5-6). *P<0.05;**P<0.01; ***P<0.001, ****P<0.0001 determined by one way ANOVA withBonferroni correction for multiple comparisons.

FIG. 5: Genetic deletion and pharmacological inhibition of IRAPattenuates Angiotensin II-mediated cardiac hypertrophy and fibrosis. (a)IRAP deficiency or IRAP inhibition prevented Ang II-mediated increase incardiac hypertrophy as assessed using cardiomyocyte cross-sectional areain Haematoxylin & Eosin (H&E) stained transverse heart sections (n=6).(b) IRAP deficiency or inhibition significantly decreased interstitialcollagen expression determined via brightfield microscopy of picrosiriusred stained transverse heart sections (n=6). Data expressed asmean±s.e.m of percentage positive stained area (n=5-6). *P<0.05;**P<0.01; ***P<0.001, ****P<0.0001 determined by one way ANOVA withBonferroni correction for multiple comparisons.

FIG. 6: Genetic deletion and pharmacological inhibition of IRAP preventsAngiotensin II-induced increase in cardiac fibrogenic markers.Representative images and quantification of positive stainedimmunofluorescence in transverse heart sections from adult (4-6 monthold) WT and IRAP^(−/−) mice treated with saline or AngII±vehicle/HFI-419 showing no change in vimentin staining (marker forfibroblast expression), decreased α-SMA expression (marker formyofibroblast expression) and decreased perivascular expression ofTGF-β₁ (fibrogenic cytokine) as well as decreased protein expression ofTGF-β1. Data expressed as mean±s.e.m of percentage positive stained areafor immunofluorescence and densitometric analysis of western blotsexpressed as relative ratio to mean of WT control±s.e.m; (n=5-6).*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 determined by one way ANOVAwith Bonferroni correction for multiple comparisons.

FIG. 7: Genetic deletion or pharmacological inhibition of IRAP preventsAngiotensin II-induced increase in cardiac reactive oxygen species(ROS), assessed by DHE staining, and inflammatory markers.Representative images and quantification of positive stainedimmunofluorescence in transverse heart sections or quantification ofprotein levels using western blot analysis from adult (4-6 month old) WTand IRAP^(−/−) mice treated with saline or Ang II±vehicle/HFI-419(n=5-6). IRAP deficiency or IRAP inhibition prevented Ang II-inducedincrease in superoxide generation, had no effect on expression of NOX-2(NADPH isoform), decreased P-IκBα expression (marker for NFκBactivation), decreased both ICAM-1 perivascular expression and totalprotein content as well as decreasing MCP-1 and macrophage (F4/80)expression. Data expressed as mean±s.e.m of percentage positive stainedarea for immunofluorescence and densitometric analysis of western blotsexpressed as relative ratio to mean of WT control±s.e.m; (n=5-6).*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 determined by one way ANOVAwith Bonferroni correction for multiple comparisons.

FIG. 8: IRAP expression is increased in aged hearts of 20 month oldwild-type (WT) mice and decreased after IRAP inhibitor treatment. (a)Representative images of IRAP expression (green) in transverse heartsections; (b) Quantification of IRAP in 5 μm thick transverse heartsections from adult (4-6 month old) and aged (18-22 month old) WT andIRAP deficient (IRAP^(−/−)) mice (n=5). (c) Quantification of IRAP in 5μm thick transverse heart sections from aged (18-22 month old) WT micetreated for 4 weeks with vehicle or the IRAP inhibitor, HFI-419 (500ng/kg/min; s.c.; n=5-8). Quantification of IRAP expressed as percentpositive stained tissue area. Data expressed as mean±s.e.m; *P<0.05,**P<0.01, determined by two way analysis of variance (ANOVA) (b) orunpaired t-test (c).

FIG. 9: IRAP deficiency prevents age-induced cardiac fibrosis. (a)Representative images of picrosirius red stained collagen in transverseheart sections of adult (4-6 month old) and aged (18-22 month old) WTand IRAP^(−/−) mice. (b) Quantification of positive stained area forinterstitial collagen, under bright field microscopy, expressed aspercent positive stained tissue area (n=5-9). Data expressed asmean±s.e.m; *P<0.05, **P<0.01 determined by two way analysis of variance(ANOVA). Analogous data for interstitial and perivascular collagenmeasured under polarized light microscopy are depicted in FIG. 10a -d.

FIG. 10: Aged IRAP deficient mice are protected against age-inducedcardiac fibrosis. Interstitial (a, b) and perivascular (c,d) collagenexpression was quantified using polarized microscopy in picrosirius redstained heart sections from young and aged WT and IRAP^(−/−) mice.Compared with bright field microscopy (FIG. 2), this analysis revealedthe same effect on collagen expression but with an absolute lower levelof collagen. Data expressed as mean±s.e.m; *P<0.05, ****P<0.0001determined by two way analysis of variance (ANOVA) for interstitialfibrosis and unpaired t-test for perivascular fibrosis; Young mice: Wt,n=8 and IRAP^(−/−), n=10; Aged mice: WT, n=14 and IRAP^(−/−), n=14).

FIG. 11: IRAP deficiency alters age-induced extracellular matrixbalance. Western blots and densitometric quantification of proteinexpression of TGF-β₁ and collagen α1 Type I (a), matrixmetalloproteinase (MMP)-2 and MMP-9 (b), MMP-8 and MMP-13 (c) in cardiactissue from aged WT and IRAP^(−/−) mice expressed as relative ratio tomean of WT control±s.e.m; (Aged mice: WT, n=4-8 and IRAP^(−/−), n=5-11).**P<0.01, determined by unpaired t-test.

FIG. 12: IRAP^(−/−) mice do not have age-induced increase in TGF-β₁ andαSMA-expressing myofibroblasts compared to WT mice. (a) Representativeimages of perivascular expression of TGF-β₁ and α-SMA-expressingmyofibroblasts via immunofluorescence staining of transverse heartsections from aged WT or IRAP^(−/−) mice. (b) Quantification of positivestained area for TGF-β₁ and α-SMA expressed as percent positive stainedtissue area (n=5-9). Data expressed as mean±s.e.m; *P<0.05, **P<0.01,****P<0.0001 determined by two way analysis of variance (ANOVA)

FIG. 13: IRAP deficiency and IRAP inhibitor treatment reducesinflammatory markers in aged mice. (a) Aged IRAP deficient micedemonstrated reduced superoxide expression (using DHE staining),decreased NFκB activation (measured via phospho-IκBα expression usingimmunofluorescence staining), reduced monocyte chemoattractant protein-1(MCP-1 via immunofluorescence), reduced macrophage expression (usingF4/80 immunofluorescence) and reduced perivascular expression ofintercellular adhesion molecule-1 (ICAM-1 via immunofluorescence) intransverse cardiac sections when compared to that seen in cardiacsections taken from aged WT mice (n=6); Data expressed as mean±s.e.m;*P<0.05; **P<0.01; ***P<0.001 determined by unpaired t-test. (b) 4 weekchronic IRAP inhibitor treatment of aged (˜20 months) WT mice reducedsuperoxide expression (using DHE staining), decreased NFκB activation(measured via phospho-IκBα expression using immunofluorescencestaining), reduced monocyte chemoattractant protein-1 (MCP-1 usingimmunofluorescence), reduced macrophage expression (using F4/80immunofluorescence) and reduced perivascular expression of intercellularadhesion molecule-1 (ICAM-1 via immunofluorescence) in transversecardiac sections when compared to that seen in cardiac sections fromaged vehicle-treated WT mice (n=6-8); Data expressed as mean±s.e.m;*P<0.05; **P<0.01; ***P<0.001 determined by unpaired t-test.

FIG. 14: Cytokine quantification was performed in hearts from aged WT(n=9) and IRAP^(−/−) (n=9) mice (a,b) and from aged vehicle—(n=6) andHFI-419—(n=9) treated mice (c,d) using Bio-Plex multiplex assay.Cytokines are grouped based on pro-inflammatory and anti-inflammatoryphenotypes with concentration of cytokines in the heart expressed asrelative ratio to aged WT control; exact fold change presented inTable 1. All data expressed as mean±s.e.m; *P<0.05; **P<0.01;***P<0.001, determined by unpaired t-test.

FIG. 15: Chronic IRAP inhibitor treatment completely reversesage-induced cardiac fibrosis. (a) Representative images of picrosiriusred stained collagen in transverse heart sections of aged (18-22 monthold) WT mice treated with vehicle or HFI-419 (500 ng/kg/min; s.c.). (b)Quantification of positive stained area for interstitial collagen, underbright field microscopy, expressed as percent positive stained tissuearea (n=5-8). Data expressed as mean±s.e.m; ****P<0.0001 determined byone way analysis of variance (ANOVA). Analogous data for interstitialcollagen measured under polarized light microscopy are depicted in FIG.16 f.

FIG. 16: Effect of chronic (4 week) pharmacological inhibition of IRAPwith HFI419 in aged mice. Chronic IRAP inhibition had no significanteffect on systolic blood pressure, SBP (a), body weight (b) and grossmeasures of cardiac hypertrophy assessed using (c) ventricular weight tobody weight ratio, VW:BW or (d) ventricular weight to tibial lengthratio, VW:TL, although age generally increased these variable comparedwith young WT mice. IRAP inhibition had no effect on cardiomyocytecross-sectional area when quantified using H&E stained heart sections(e), while IRAP inhibition significantly decreased interstitial collagenexpression to those levels observed in young WT mice (f), determined viapolarized microscopy of picrosirius red stained heart cross-sections.Aged vehicle-treated mice: n=10 and aged HFI-419-treated mice, n=10;Data expressed as mean±s.e.m; *P<0.05, **P<0.001, determined by one-wayANOVA.

FIG. 17: Chronic IRAP inhibitor treatment alters age-inducedextracellular matrix balance. Western blots and densitometricquantification of protein expression of precursor and mature collagen,matrix metalloproteinase (MMP)-2, MMP-8, MMP-9, MMP-13 and TIMP-1 incardiac tissue from aged vehicle and HFI-419 treated WT mice expressedas relative ratio to mean of vehicle-treated WT control±s.e.m; (n=4 inall groups). *P<0.05, **P<0.01, determined by unpaired t-test.

FIG. 18: Chronic IRAP inhibition with HFI-419 in aged WT micesignificantly decreased levels of TGF-β₁ and α-SMA-expressingmyofibroblasts compared to vehicle-treated aged WT mice. Quantificationof positive stained area for TGF-β₁ and α-SMA expressed as percentpositive stained tissue area (n=5-8). Data expressed as mean±s.e.m;**P<0.01, ****P<0.0001 determined by one way analysis of variance(ANOVA).

FIG. 19: Effect of two structurally distinct IRAP inhibitors to reverseage-induced cardiac fibrosis. Aged (20 month old) WT mice werechronically treated with vehicle, compound 1 (denoted as Class 1) orcompound 2 (denoted Class 2) for 4 weeks. Picrosirius red staining oftransverse heart sections from each of these groups demonstrated clearreversal of age-induced cardiac fibrosis (n=3). Data expressed asmean±s.e.m of percentage positive stained area. *P<0.05; determined byone way ANOVA with Bonferroni correction for multiple comparisons.

FIG. 20: Genetic deletion and pharmacological inhibition of IRAP improveheart function and decrease infarct area following ischemic-reperfusion(I/R) injury. Heart function measurements were performed using theisolated Langendorff heart preparation with a 40 minute ischaemic/1 hourreperfusion injury (IR, ischaemic reperfusion). Hearts were stopped indiastole by placing in high potassium solution (PSS; 100 mM) for 3minutes, after which they were sliced and stained with TTZ.Representative images showing infarct area from each group are shown in(a). Infarct area appears white and is outlined within the dotted lineregion. Infarct area is quantified as percentage stained area acrossboth superior and inferior surfaces of 5-7 heart slices from young WT(n=7), aged IRAP^(−/−) (n=10), aged vehicle-treated (n=8) or HFI-419treated (n=8) WT mice. Data expressed as mean±s.e.m, **P<0.01 determinedby one way ANOVA. IRAP deficiency or chronic IRAP inhibition improvedrecovery of left ventricular developed pressure (LVDP) (b), rate of leftventricular contraction (+dp/dt) (c) and rate of left ventricularrelaxation (−dp/dt) (d) following ischemic injury. Data expressed asmean±s.e.m. *P<0.05, **P<0.01 was determined using two-way ANOVA withpost hoc Bonferroni test on LVDP and ±dp/dt. Echocardiography studieswere performed in aged (˜22 month old) WT and IRAP−/− mice with cardiacfunction compared to that of young (3 month old) WT mice. Aged WT mice(n=5) had a significant reduction in left ventricular ejection fraction(LVEF) (e) and a trend towards reduced LV contractility (f) compared toyoung WT mice (n=5) with aged IRAP−/− mice (n=4) protected against theseage-induced changes in cardiac function. Data expressed as mean±s.e.m,**P<0.01 determined by one way ANOVA.

FIG. 21: Phenotypic differences between WT and IRAP deficient mice at 6months and ˜22 months of age. There was minimal effect of age andgenotype on systolic blood pressure, SBP when compared at young (˜5months old) and aged (˜20 months old) time points (a). As expected,there were increases in body weight of WT and IRAP^(−/−) mice associatedwith aging (b). Gross measures of cardiac hypertrophy using (c)ventricular weight to body weight ratio, VW:BW or (d) ventricular weightto tibial length ratio, VW:TL, found a significant effect of aging toincrease the VW:BW ratio in IRAP^(−/−) mice as well as the VW:TL ratioin both strains that was largely independent of genotype. Cardiachypertrophy was further assessed using cross-sectional cardiomyocytearea measurement. Representative images of cardiomyocytes in H&E-stainedheart sections are shown in (e) with quantification of cardiomyocytecross-sectional area performed in 6 fields of view per heart section(f). Data are expressed as mean±s.e.m; *P<0.05, **P<0.01, ***P<0.001,****P<0.0001 determined by two way analysis of variance (ANOVA) (Youngmice: Wt, n=8 and IRAP^(−/−), n=10; Aged mice: WT, n=16 and IRAP^(−/−),n=16).

FIG. 22: IRAP expression is increased in kidneys from aged (˜20 monthold) WT mice and decreased after pharmacological inhibition with an IRAPinhibitor. (a) Quantification of IRAP expression in 5 μm thick coronalkidney sections from adult (4-6 month old) and aged (18-22 month old) WTand IRAP^(−/−) mice (n=4). (b) Quantification of IRAP expression in 5 μmthick coronal kidney sections from aged (18-22 month old) WT micetreated for 4 weeks with vehicle or HFI-419 (500 ng/kg/min; s.c.; n=4).IRAP inhibitor treatment tended to decrease IRAP expression compared tovehicle-treated aged controls. Quantification of IRAP expressed aspercent positive stained tissue area. Data expressed as mean±s.e.m;*P<0.05 determined by one way analysis of variance (ANOVA) (a) orunpaired t-test (b).

FIG. 23: Effect of IRAP deficiency or IRAP inhibition on development ofage-induced kidney fibrosis. (a) Representative images andquantification of picrosirius red stained interstitial collagen incoronal kidney sections of adult (4-6 month old) and aged (18-22 monthold) WT and aged IRAP^(−/−) mice demonstrating IRAP deficiency preventsage-induced increase in interstitial kidney fibrosis (n=4). (b)Representative images and quantification of picrosirius red stainedinterstitial collagen in coronal kidney sections of aged (18-22 monthold) vehicle and HFI-419 treated WT mice demonstrating IRAP inhibitionreverses age-induced increase in interstitial kidney fibrosis (n=4).Data expressed as percent positive stained tissue area. Data expressedas mean±s.e.m; *P<0.05, **P<0.01, ***P<0.001 determined by one wayanalysis of variance (ANOVA) (a) or unpaired t-test (b).

FIG. 24: IRAP deficiency and IRAP inhibition prevent or reverse,respectively, age-induced increase in α-SMA-expressing myofibroblastscompared to age-matched controls. (a) Quantification of positive stainedarea for α-SMA-expressing myofibroblasts via immunofluorescence stainingof coronal kidney sections from adult (4-6 month old) and aged (18-22month old) WT and aged IRAP^(−/−) mice. α-SMA expressed as percentpositive stained tissue area with data expressed as mean±s.e.m (n=4);****P<0.0001 determined by one way analysis of variance (ANOVA). (b)Quantification of positive stained area for α-SMA-expressingmyofibroblasts via immunofluorescence staining of coronal kidneysections from aged (18-22 month old) vehicle and HFI-419 treated WTmice. α-SMA expressed as percent positive stained tissue area with dataexpressed as mean±s.e.m (n=4).

FIG. 25: Increased IRAP expression in human cardiac fibroblastsstimulated with Angiotensin II. Representative images showing primaryhuman cardiac fibroblasts stimulated with increasing concentrations ofAng II induced an increase in expression of IRAP.

FIG. 26: IRAP inhibitor dose-dependently decreased α-SMA and collagenexpression in human cardiac fibroblasts. (a) Representative imagesshowing increased expression of α-SMA (red; marker for myofibroblasts)and collagen (green) when human cardiac fibroblasts (HCFs) werestimulated with Ang II (0.1 μM). Combined Ang II and HFI-419 treatment(0.01 to 1 μM) decreased α-SMA and collagen expression. (b) Quantitativedata from western blots confirming dose-dependent decrease in proteinexpression of α-SMA and collagen when HCFs were co-treated with AngII+increasing concentrations of HFI-419 (n=10-12). Data expressed asmean±s.e.m; densitometric analysis of western blots expressed asrelative ratio to mean of control cells±s.e.m; *P<0.05; **P<0.01;***P<0.001 determined by one way ANOVA with Bonferroni correction formultiple comparisons.

FIG. 27: Liver sections from WT (top panels) and IRAP KO (bottom panels)mice stained with OilRedO to indicate steatosis. The liver sections fromWT mice displayed greater macrovesicular steatosis indicated by thearrows.

FIG. 28: Chronic IRAP inhibitor treatment reverses HSD-induced liverfibrosis. (a) Representative images of masson trichrome stained collagenin liver sections of WT mice treated normal diet (ND) or high salt diet(HSD)+vehicle or HFI-419 (500 ng/kg/min; s.c.). (b) Quantification ofpositive stained area for collagen, under bright field microscopy,expressed as percent positive stained tissue area (n=3). Data expressedas mean±s.e.m; **P<0.01 determined by one way analysis of variance(ANOVA).

DETAILED DESCRIPTION OF THE EMBODIMENTS

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

Reference will now be made in detail to certain embodiments of theinvention. While the invention will be described in conjunction with theembodiments, it will be understood that the intention is not to limitthe invention to those embodiments. On the contrary, the invention isintended to cover all alternatives, modifications, and equivalents,which may be included within the scope of the present invention asdefined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described. It will be understoodthat the invention disclosed and defined in this specification extendsto all alternative combinations of two or more of the individualfeatures mentioned or evident from the text or drawings. All of thesedifferent combinations constitute various alternative aspects of theinvention.

All of the patents and publications referred to herein are incorporatedby reference in their entirety.

For purposes of interpreting this specification, terms used in thesingular will also include the plural and vice versa.

The inventors have identified the enzyme, insulin regulatedaminopeptidase (IRAP; also known as the angiotensin subtype 4receptor—AT₄R, placental leucine aminopeptidase or oxytocinase) as anovel target to combat fibrosis. It is proposed that Ang IV binds toIRAP and acts to inhibit the catalytic activity of this enzyme, howeveras yet there are no chronic studies exploring the potential benefits ofIRAP inhibition in the context of cardiovascular disease. The inventorshypothesized that removal or blockade of IRAP activity would protectagainst age-mediated increases in cardiac fibrosis and inflammation, orother cardiovascular disease-related or tissue injury related organfibrosis, to improve cardiac and vascular function. The inventors testedthis hypothesis in (i) a prevention model of aged male WT and IRAP^(−/−)mice (18-22 month old), (ii) a prevention model using Ang II infusion toinduce fibrosis and inflammation in multiple organs, and in (iii) anintervention model by administering a small molecule inhibitor of IRAPto aged WT mice with established cardiovascular pathologies, in order toreverse CVD. The inventors found that IRAP deficiency or pharmacologicalinhibition of IRAP protected against and, more importantly, reversedage- or injury-induced organ fibrosis (e.g. in heart and kidneys) to thelevel exhibited in young mice, in part by inhibiting synthesis andenhancing degradation of collagen. In addition, IRAP inhibitiondecreased cardiac ROS (reactive oxygen species) and inflammatorymediators downstream of NFκB, collectively pushing towards ananti-inflammatory phenotype thus contributing to overall cardiac andvascular improvement in aging. A similar anti-fibrotic andanti-inflammatory phenotype was also shown in IRAP−/− mice and bypharmacological IRAP inhibition in mice treated with Ang II to inducecardiovascular pathologies such as organ fibrosis and inflammation.

The present invention is based on results described herein whereinhibition of IRAP was confirmed as having a role in fibrotic disease,particularly age-induced fibrotic disease, using IRAP deficient mice orpharmacological inhibition with an IRAP inhibitor. The inventorsdemonstrated that IRAP-deficient mice were protected from fibrosis andfurther, that those mice with experimentally induced or age relatedfibrosis that were administered an IRAP inhibitor were successfullytreated for fibrosis, as demonstrated by a consistent ability of IRAPinhibitors to reduce fibrosis and the expression of fibrogenicmediators.

An advantage of the invention is the surprising finding that treatmentwith an inhibitor of IRAP at the time of established fibrotic diseaseleads to a reversal of fibrosis. Pharmacological inhibition of IRAPtherefore not only has the effect of halting progression of fibrosis,such as age- or injury-induced fibrosis, but reversing the existingsymptoms, such as collagen deposition. The invention therefore findsparticular application to subjects that are diagnosed with fibrosis,such as age-induced fibrosis or for cardiovascular diseases that areoften associated with organ fibrosis. Further, reversing the hallmarksof age-induced fibrosis indicates that the invention can be applied tosubjects with advanced fibrosis.

As used herein, an “IRAP inhibitor” or “inhibitor of IRAP” is anycompound that inhibits the activity of IRAP (IRAP; also known as theangiotensin subtype 4 receptor —AT₄R, placental leucine aminopeptidaseor oxytocinase). Inhibition of activity of IRAP may also include areduction in the level or amount of IRAP protein, RNA or DNA in a cell.The compound may be a competitive, non-competitive, orthosteric,allosteric, or partial inhibitor. In a preferred form the compound is amolecule that inhibits the enzyme activity of IRAP for example bybinding the active site, or competing with the enzyme substrate orco-effector or signalling mechanism. In a preferred form the compound isa molecule that inhibits the activity of IRAP by disrupting thesignalasome or any other protein-protein interaction required for theactivity of IRAP.

The inhibitor may be specific for IRAP and only have some low levelinhibitory activity against other receptors (for example, a Ki ofgreater than about 50 μM or 1001 μM, preferably 1 mM against otherreceptors as measured using an assay as described herein, or for examplea Ki against other receptors at least 10× greater than the Ki againstIRAP). Preferably, the inhibitor of IRAP is a substance that limits theactivity of IRAP to 10% or less in comparison with control. Control is asolvent, in which the inhibitor is tested, used at the same quantity,however, without the inhibitor. The enzymatic activities of IRAP may bedetermined by the hydrolysis of the synthetic substrate Leu-MCA(Sigma-Aldrich, Missouri, USA) monitored by the release of a fluorogenicproduct, MCA, at excitation and emission wavelengths of 380 and 440 nm,respectively according to Albiston et al. 2008 The FASEB Journal22:4209-4217 or other method described herein. In preferred forms, theinhibitor may be a small molecule chemical compound or interfering RNA(e.g. siRNA). The inhibitor may also be an antibody such as a monoclonalantibody.

Preferably, an antibody inhibitor is a neutralising antibody inhibitor.

The term “small molecule” denotes a generally low molecular weightcompound and includes organic and inorganic compounds. In general, asmall molecule has a well-defined chemical formula with a singlemolecular weight. Preferably, a small molecule has a molecular weight ofless than 3000 daltons. More preferably, a small molecule has amolecular weight of less than 2000 daltons. In some embodiments of thisinvention, the small molecule has a molecular weight of less than 1000daltons. Some non-limiting examples of small molecules include lipidssuch as fatty acids; saccharides (mono, di or poly); xenobiotics;organometallic compounds and natural products.

The inhibitor of IRAP may exhibit a Ki value of less than 1 mM,preferably less than 100 μM, more preferably less than 101 μM, asdetermined by an assay as described herein, for example ofaminopeptidase activity or substrate degradation. Typically, the assayof amino peptidase activity comprises hydrolysis of the syntheticsubstrate L-Leucine 7-amido-4-methyl coumarin hydrochloride (Leu-MCA)monitored by release of the fluorogenic product MCA. The assay ofsubstrate degradation may be degradation of the peptide substratesCYFQNCPRG (SEQ ID NO: 1), CYIQNCPLG—NH2 (SEQ ID NO: 6) or YGGFL (SEQ IDNO: 2).

Inhibitors of IRAP are known in the art. For example, IRAP inhibitorsdescribed in Albiston et al. (2008) The FASEB Journal 22:4209-4217;Albiston et al. (2011), British Journal of Pharmacology, 164:37-47,Albiston, et al. J. Biol. Chem. 276, 48263-48266; U.S. Pat. No.6,066,672; Albiston, et al. Pharmacol. Ther. 116, 417-427; Axen, et al.(2006) J. Pept. Sci. 12, 705-713; Albiston et al. (2010) MolecularPharmacology, 78(4): 600-607; Mountford, et al. (2014) J Med Chem 57(4):1368-1377; Andersson et al. J Med Chem (2010) 53, 8059, Andersson et al.(2011) J Med Chem 54(11):3779-3792; WO2009065169; WO2010001079; WO2000/012544; US 2004/0086510; WO 2003/011304; and WO2006026832, and maybe useful in the present invention.

An inhibitor of IRAP as described herein may have a structure accordingto Formula (I):

wherein

-   -   A is aryl, heteroaryl carbocyclyl or heterocyclyl, each of which        may be optionally substituted, when R¹ is NHCOR₈;        -   or quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl,            quinoxalinyl, 1,8-naphthyridyl, phthalazinyl or pteridinyl,            each of which may be optionally substituted, when R¹ is            NR₇R₈, NHCOR₈, N(COR₈)₂, N(COR₇)(COR₈), N═CHOR₈ or N═CHR₈;    -   X is O, NR′ or S, wherein R′ is hydrogen, optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        alkynyl, optionally substituted aryl, optionally substituted        acyl, optionally substituted heteroaryl, optionally substituted        carbocyclyl or optionally substituted heterocyclyl;    -   R₇ and R₈ are independently selected from hydrogen, optionally        substituted alkyl, optionally substituted aryl, or R₇ and R₈,        together with the nitrogen atom to which they are attached form        a 3-8-membered ring which may be optionally substituted;        -   R² is CN, CO₂R⁹, C(O)O(O)R⁹, C(O)R⁹ or C(O)NR⁹R¹⁰ wherein R⁹            and R¹⁰ are independently selected from alkyl, alkenyl,            alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, each            of which may be optionally substituted, and hydrogen; or R⁹            and R^(o1) together with the nitrogen atom to which they are            attached, form a 3-8-membered ring which may be optionally            substituted;    -   R₃-R₆ are independently selected from hydrogen, halo, nitro,        cyano alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl,        carbocyclyl, hydroxy, alkoxy, alkenyloxy, alkynyloxy,        alkynyloxy, aryloxy, heteroaryloxy, heterocyclyloxy, amino,        acyl, acyloxy, carboxy, carboxyester, methylenedioxy, amido,        thio, alkylthio, alkenylthio, alkynylthio, arylthio,        heteroarylthio, heterocyclylthio, carbocyclylthio, acylthio and        azido, each of which may be optionally substituted where        appropriate, or any two adjacent R³-R⁶, together with the atoms        to which they are attached, form a 3-8-membered ring which may        be optionally substituted; and        -   Y is hydrogen or C₁₋₁₀alkyl,

or a pharmaceutically acceptable salt or solvate thereof.

In one preferred embodiment, A is optionally substituted heteroaryl whenR¹ is NHCOR₈. More preferably, A is pyridinyl.

In another preferred embodiment, X is O.

In yet another preferred embodiment, R² is CO₂R⁹.

In one preferred embodiment, R₅ is hydroxyl.

In one embodiment, the inhibitor has the structure:

An inhibitor of IRAP as described herein may have a structure accordingto Formula (II):

wherein

-   -   A is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl,        heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each        of which may be optionally substituted;    -   R_(A) and R_(B) are independently selected from hydrogen, alkyl        and acyl;    -   R₁ is selected from CN or CO₂R_(C);    -   R₂ is selected from CO₂R_(C) and acyl;    -   R₃ is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl,        heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each        of which may be optionally substituted; or    -   R₂ and R₃ together form a 5-6-membered saturated        keto-carbocyclic ring:

-   -   -   wherein n is 1 or 2;        -   and which ring may be optionally substituted one or more            times by C₁₋₆alkyl; or

    -   R₂ and R₃ together form a 5-membered lactone ring (a) or a        6-membered lactone ring (b)

-   -   -   wherein            is an optional double bond and R′ is alkyl.

    -   R_(C) is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl,        heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each        of which may be optionally substituted;

or a pharmaceutically acceptable salt, solvate or prodrug thereof.

In a preferred embodiment, A is optionally substituted aryl. Morepreferably, A is aryl substituted with —COOH, or a salt, ester orprodrug thereof. For example, A may be aryl substituted with —CO₂—NH₄ ⁺.In another preferred embodiment, R₁ is CN.In yet another preferred embodiment, R₂ is acyl.In one embodiment, the inhibitor has the structure:

In other embodiments, the inhibitor has a structure selected from thegroup consisting of:

and/or a pharmaceutically acceptable salt, solvate or prodrug thereof.

In another embodiment of the present invention, the inhibitor has astructure according to Formula (III):

wherein

-   -   R₁ is H or CH₂COOH; and    -   n is 0 or 1; and    -   m is 1 or 2; and    -   W is CH or N;

or a pharmaceutically acceptable salt, solvate or prodrug thereof.

In one embodiment, the inhibitor has the structure:

In another embodiment of the present invention, the inhibitor has astructure according to any one of the following sequences:

(SEQ ID NO: 3) Val-Tyr-Ile-His-Pro-Phe, (SEQ ID NO: 4)c[Cys-Tyr-Cys]-His-Pro-Phe, and (SEQ ID NO: 5)c[Hcy-Tyr-Hcy]-His-Pro-Phe.

In yet another embodiment of the present invention, the inhibitor has astructure according to the compound

As used herein, the term “alkyl” or “alk”, used either alone or incompound words denotes straight chain, or branched alkyl, preferablyC₁₋₂₀ alkyl, e.g. C₁₋₁₀ or C₁₋₆. Examples of straight chain and branchedalkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,t-butyl, n-pentyl, 1,2-dimethylpropyl, 1,1-dimethyl-propyl, hexyl,4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl,1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl,1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2,-trimethylpropyl,1,1,2-trimethylpropyl, heptyl, 5-methylhexyl, 1-methylhexyl,2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl,1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethyl-pentyl,1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl,6-methylheptyl, 1-methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-,3-, 4-, 5-, 6- or 7-methyl-octyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-,2- or 3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and8-methylnonyl, 1-, 2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or4-propylheptyl, undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-ethylnonyl, 1-, 2-, 3-, 4- or5-propylocytl, 1-, 2- or 3-butylheptyl, 1-pentylhexyl, dodecyl, 1-, 2-,3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-,7- or 8-ethyldecyl, 1-, 2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or4-butyloctyl, 1-2-pentylheptyl and the like. Where an alkyl group isreferred to generally as “propyl”, butyl” etc, it will be understoodthat this can refer to any of straight or branched isomers whereappropriate.

An alkyl group may be optionally substituted by one or more optionalsubstituents as herein defined.

The term “alkenyl” as used herein denotes groups formed from straightchain or branched hydrocarbon residues containing at least one carbon tocarbon double bond including ethylenically mono-, di- orpoly-unsaturated alkyl groups as previously defined, preferably C₂₋₂₀alkenyl (e.g. C₂₋₁₀ or C₂₋₆). Examples of alkenyl include vinyl, allyl,1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl,1-hexenyl, 3-hexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, 1-nonenyl,2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl,1-4,pentadienyl, 1,3-hexadienyl and 1,4-hexadienyl. An alkenyl group maybe optionally substituted by one or more optional substituents as hereindefined.

As used herein the term “alkynyl” denotes groups formed from straightchain or branched hydrocarbon residues containing at least onecarbon-carbon triple bond including ethynically mono-, di- orpoly-unsaturated alkyl groups as previously defined. Unless the numberof carbon atoms is specified the term preferably refers to C₂₋₂₀ alkynyl(e.g. C₂₋₁₀ or C₂₋₆). Examples include ethynyl, 1-propynyl, 2-propynyl,and butynyl isomers, and pentynyl isomers. An alkynyl group may beoptionally substituted by one or more optional substituents as hereindefined.

Terms written as “[group]oxy” refer to a particular group when linked byoxygen, for example, the terms “alkoxy”, “alkenoxy”, “alkynoxy”,“aryloxy” and “acyloxy” respectively denote alkyl, alkenyl, alkynyl,aryl and acyl groups as hereinbefore defined when linked by an oxygenatom. Terms written as “[group]thio” refer to a particular group whenlinked by sulfur, for example, the terms “alkylthio”, “alkenylthio”,alkynylthio” and “arylthio” respectively denote alkyl, alkenyl, alkynyl,aryl groups as hereinbefore defined when linked by a sulfur atom.Similarly, a term written as “[groupA]groupB” is intended to refer to agroupA when linked by a divalent form of groupB, for example,“hydroxyalkyl” is a hydroxy group when linked by an alkylene group.

The term “halogen” (“halo”) denotes fluorine, chlorine, bromine oriodine (fluoro, chloro, bromo or iodo).

The term “aryl” (or “carboaryl)”, or the abbreviated form “ar” used incompound words such as “aralkyl”, denotes any of mono-, bi- orpolcyclic, (including conjugated and fused) hydrocarbon ring systemscontaining an aromatic residue. Examples of aryl include phenyl,biphenyl, terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl(tetralinyl), anthracenyl, dihydroanthracenyl, benzanthracenyl,dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, idenyl,isoindenyl, indanyl, azulenyl and chrysenyl. Particular examples of arylinclude phenyl and naphthyl. An aryl group may be optionally substitutedby one or more optional substituents as herein defined.

The term “carbocyclyl” includes any of non-aromatic monocyclic, bicyclicand polycyclic, (including fused, bridged or conjugated) hydrocarbonresidues, e.g. C₃₋₂₀ (such as C₃₋₁₀, C₃₋₈ or C₅₋₆). The rings may besaturated, for example cycloalkyl, or may possess one or more doublebonds (cycloalkenyl) and/or one or more triple bonds (cycloalkynyl).Examples of particular carbocyclyl are monocyclic 5-6-membered orbicyclic 9-10 membered ring systems. Suitable examples includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl,cyclooctenyl, cyclopentadienyl, cyclohexadienyl, cyclooctatetraenyl anddecalinyl. A carbocyclyl group may be optionally substituted by one ormore optional substituents as herein defined. In particular, amonocarbocyclyl group may be substituted by a bridging group to form abicyclic bridged group.

The term “carbocyclyl” includes any of non-aromatic monocyclic, bicyclicand polycyclic, (including fused, bridged or conjugated) hydrocarbonresidues, e.g. C₃₋₂₀ (such as C₃₋₁₀, C₃₋₈ or C₅₋₆). The rings may besaturated, for example cycloalkyl, or may possess one or more doublebonds (cycloalkenyl) and/or one or more triple bonds (cycloalkynyl).Examples of carbocyclyl include monocyclic 5-6-membered or bicyclic 9-10membered ring systems. Suitable examples include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl, cyclooctenyl,cyclopentadienyl, cyclohexadienyl, cyclooctatetraenyl and decalinyl. Acarbocyclyl group may be optionally substituted by one or more optionalsubstituents as herein defined. A monocarbocyclyl group may besubstituted by a bridging group to form a bicyclic bridged group.

The term “heterocyclyl” when used alone or in compound words includesany of monocyclic, bicyclic or polycyclic, (including fuse, bridged orconjugated) hydrocarbon residues, such as C₃₋₂₀ (e.g. C₃₋₁₀ or C₃₋₈)wherein one or more carbon atoms are independently replaced by aheteroatom so as to provide a group containing a non-aromatic heteroatomcontaining ring. Suitable heteroatoms include, O, N, S, P and Se,particularly O, N and S. Where two or more carbon atoms are replaced,this may be by two or more of the same heteroatom or by differentheteroatoms. The heterocyclyl group may be saturated or partiallyunsaturated, e.g. possess one or more double bonds. Particularlypreferred heterocyclyl are monocyclic 5-6- and bicyclic 9-10-memberedheterocyclyl. Examples of heterocyclyl groups may include azridinyl,oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 2H-pyrrolyl,pyrrolidinyl, 1-, 2- and 3-pyrrolinyl, piperidyl, piperazinyl,morpholinyl, indolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,thiomorpholinyl, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyrrolyl, tetrahydrothiophenyl (tetramethylene sulfide),pyrazolinyl, dioxalanyl, thiazolidinyl, isoxazolidinyl, dihydropyranyl,oxazinyl, thiazinyl, thiomorpholinyl, oxathianyl, dithianyl, trioxanyl,thiadiazinyl, dithiazinyl, trithianyl, azepinyl, oxepinyl, thiepinyl,indenyl, indanyl, 3H-indolyl, isoindolinyl, 4H-quinolazinyl, chromenyl,chromanyl, isochromanyl, benzoxazinyl (2H-1,3, 2H-1,4-, 1H-2,3-,4H-3,1-4H-1,4) pyranyl and dihydropyranyl. A heterocyclyl group may beoptionally substituted by one or more optional substituents as definedherein.

The term “heteroaryl” includes any of monocyclic, bicyclic, polycyclic,fused, bridged or conjugated hydrocarbon residues, wherein one or morecarbon atoms are replaced by a heteroatom so as to provide a residuehaving at least one aromatic heteroatom-containing ring. Exemplaryheteroaryl have 3-20 ring atoms, e.g. 3-10. Particularly preferredheteroaryl are 5-6 monocyclic and 9-10 membered bicyclic ring systems.Suitable heteroatoms include, O, N, S, P and Se, particularly O, N andS. Where two or more carbon atoms are replaced, this may be by two ormore of the same heteroatom or by different heteroatoms. Suitableexamples of heteroaryl groups may include pyridyl, pyrrolyl, thienyl,imidazolyl, furanyl, benzothienyl, isobenzothienyl, benzofuranyl,isobenzofuranyl, indolyl, isoindolyl, pyrazolyl, pyrazinyl, pyrimidinyl,pyridazinyl, indolizinyl, quinolyl, isoquinolyl, phthalazinyl,1,5-naphthyridinyl, quinozalinyl, quinazolinyl, quinolinyl, oxazolyl,thiazolyl, isothiazolyl, isoxazolyl, triazolyl, oxadialzolyl,oxatriazolyl, triazinyl, tetrazolyl and furazanyl. A heteroaryl groupmay be optionally substituted by one or more optional substituents asdefined herein.

The term “acyl” either alone or in compound words denotes a groupcontaining the moiety C═O. In some embodiments acyl does not include acarboxylic acid, ester or amide. Acyl includes C(O)—Z, wherein Z ishydrogen or an alkyl, aryl, heteroaryl, carbocyclyl, heterocyclyl,arylalkyl, heteroarylalkyl, carbocyclylalkyl, or heterocyclylalkylresidue. Examples of acyl include formyl, straight chain or branchedalkanoyl (e.g. C₁₋₂₀) such as, acetyl, propanoyl, butanoyl,2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl,heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl,tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl,octadecanoyl, nonadecanoyl and icosanoyl; cycloalkylcarbonyl such ascyclopropylcarbonyl cyclobutylcarbonyl, cyclopentylcarbonyl andcyclohexylcarbonyl; aroyl such as benzoyl, toluoyl and naphthoyl;aralkanoyl such as phenylalkanoyl (e.g. phenylacetyl, phenylpropanoyl,phenylbutanoyl, phenylisobutylyl, phenylpentanoyl and phenylhexanoyl)and naphthylalkanoyl (e.g. naphthylacetyl, naphthylpropanoyl andnaphthylbutanoyl]; aralkenoyl such as phenylalkenoyl (e.g.phenylpropenoyl, phenylbutenoyl, phenylmethacryloyl, phenylpentenoyl andphenylhexenoyl and naphthylalkenoyl (e.g. naphthylpropenoyl,naphthylbutenoyl and naphthylpentenoyl); aryloxyalkanoyl such asphenoxyacetyl and phenoxypropionyl; arylthiocarbamoyl such asphenylthiocarbamoyl; arylglyoxyloyl such as phenylglyoxyloyl andnaphthylglyoxyloyl; arylsulfonyl such as phenylsulfonyl andnapthylsulfonyl; heterocycliccarbonyl; heterocyclicalkanoyl such asthienylacetyl, thienylpropanoyl, thienylbutanoyl, thienylpentanoyl,thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl andtetrazolylacetyl; heterocyclicalkenoyl such as heterocyclicpropenoyl,heterocyclicbutenoyl, heterocyclicpentenoyl and heterocyclichexenoyl;and heterocyclicglyoxyloyl such as thiazolyglyoxyloyl andthienylglyoxyloyl. The R and Z residues may be optionally substituted asdescribed herein.

In this specification “optionally substituted” is taken to mean that agroup may be unsubstituted or further substituted or fused (so as toform a condensed bi- or polycyclic group) with one, two, three or moreof organic and inorganic groups, including those selected from: alkyl,alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, heteroaryl, acyl,aralkyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl,alkylcarbocyclyl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl,halocarbocyclyl, haloheterocyclyl, haloheteroaryl, haloacyl,haloaryalkyl, hydroxy, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl,hydroxycarbocyclyl, hydroxyaryl, hydroxyheterocyclyl, hydroxyheteroaryl,hydroxyacyl, hydroxyaralkyl, alkoxyalkyl, alkoxyalkenyl, alkoxyalkynyl,alkoxycarbocyclyl, alkoxyaryl, alkoxyheterocyclyl, alkoxyheteroaryl,alkoxyacyl, alkoxyaralkyl, alkoxy, alkenyloxy, alkynyloxy, aryloxy,carbocyclyloxy, aralkyloxy, heteroaryloxy, heterocyclyloxy, acyloxy,haloalkoxy, haloalkenyloxy, haloalkynyloxy, haloaryloxy,halocarbocyclyloxy, haloaralkyloxy, haloheteroaryloxy,haloheterocyclyloxy, haloacyloxy, nitro, nitroalkyl, nitroalkenyl,nitroalkynyl, nitroaryl, nitroheterocyclyl, nitroheteroaryl,nitrocarbocyclyl, nitroacyl, nitroaralkyl, amino (NH₂), alkylamino,dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino,aralkylamino, diaralkylamino, acylamino, diacylamino, heterocyclamino,heteroarylamino, carboxy, carboxyester, amido, alkylsulphonyloxy,arylsulphenyloxy, alkylsulphenyl, arylsulphenyl, thio, alkylthio,alkenylthio, alkynylthio, arylthio, aralkylthio, carbocyclylthio,heterocyclylthio, heteroarylthio, acylthio, sulfoxide, sulfonyl,sulfonamido, aminoalkyl, aminoalkenyl, aminoalkynyl, aminocarbocyclyl,aminoaryl, aminoheterocyclyl, aminoheteroaryl, aminoacyl, aminoaralkyl,thioalkyl, thioalkenyl, thioalkynyl, thiocarbocyclyl, thioaryl,thioheterocyclyl, thioheteroaryl, thioacyl, thioaralkyl, carboxyalkyl,carboxyalkenyl, carboxyalkynyl, carboxycarbocyclyl, carboxyaryl,carboxyheterocyclyl, carboxyheteroaryl, carboxyacyl, carboxyaralkyl,carboxyesteralkyl, carboxyesteralkenyl, carboxyesteralkynyl,carboxyestercarbocyclyl, carboxyesteraryl, carboxyesterheterocyclyl,carboxyesterheteroaryl, carboxyesteracyl, carboxyesteraralkyl,amidoalkyl, amidoalkenyl, amidoalkynyl, amidocarbocyclyl, amidoaryl,amidoheterocyclyl, amidoheteroaryl, amidoacyl, amidoaralkyl,formylalkyl, formylalkenyl, formylalkynyl, formylcarbocyclyl,formylaryl, formylheterocyclyl, formylheteroaryl, formylacyl,formylaralkyl, acylalkyl, acylalkenyl, acylalkynyl, acylcarbocyclyl,acylaryl, acylheterocyclyl, acylheteroaryl, acylacyl, acylaralkyl,sulfoxidealkyl, sulfoxidealkenyl, sulfoxidealkynyl,sulfoxidecarbocyclyl, sulfoxidearyl, sulfoxideheterocyclyl,sulfoxideheteroaryl, sulfoxideacyl, sulfoxidearalkyl, sulfonylalkyl,sulfonylalkenyl, sulfonylalkynyl, sulfonylcarbocyclyl, sulfonylaryl,sulfonylheterocyclyl, sulfonylheteroaryl, sulfonylacyl, sulfonylaralkyl,sulfonamidoalkyl, sulfonamidoalkenyl, sulfonamidoalkynyl,sulfonamidocarbocyclyl, sulfonamidoaryl, sulfonamidoheterocyclyl,sulfonamidoheteroaryl, sulfonamidoacyl, sulfonamidoaralkyl, nitroalkyl,nitroalkenyl, nitroalkynyl, nitrocarbocyclyl, nitroaryl,nitroheterocyclyl, nitroheteroaryl, nitroacyl, nitroaralkyl, cyano,sulfate, sulfonate, phosphonate and phosphate groups. Optionalsubstitution may also be taken to refer to where a CH₂ group in a chainor ring is replaced by a carbonyl group (C═O) or a thiocarbonyl group(C═S), where 2 adjacent or non-adjacent carbon atoms (e.g. 1,2- or 1,3)are substituted by one end each of a —O—(CH₂)S—O— or —NRX—(CH₂)S—NRX—group, wherein s is 1 or 2 and each R^(x) is independently H orC₁₋₆alkyl, and where 2 adjacent or non-adjacent atoms, independentlyselected from C and N, are substituted by one end each of a C₁₋₅alkyleneor C₂₋₅alkenylene group (so as to form a bridged group).

Exemplary optional substituents include those selected from: alkyl,(e.g. C₁₋₆alkyl such as methyl, ethyl, propyl, butyl), cycloalkyl (e.g.C₃₋₆cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl), hydroxyalkyl (e.g. hydroxyC₁₋₆alkyl, such as hydroxymethyl,hydroxyethyl, hydroxypropyl), alkoxyalkyl (e.g. C₁₋₆alkoxyC₁₋₆alkyl,such as methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl,ethoxyethyl, ethoxypropyl), alkoxy (e.g. C₁₋₆alkoxy, such as methoxy,ethoxy, propoxy, butoxy), alkoxyalkoxy (e.g. C₁₋₆alkoxyC₁₋₆alkoxy, suchas methoxymethoxy, methoxyethoxy, methoxypropoxy, ethoxymethoxy,ethoxyethoxy, ethoxypropoxy, propoxymethoxy, propoxyethoxy,propoxypropoxy) cycloalkoxy (e.g. cyclopropoxy, cyclobutoxy,cyclopentoxyl, cyclohexyloxy), halo, haloalkyl(e.g. haloC₁₋₆alkyl, suchas chloromethyl, difluoromethyl, trifluoromethyl, trichloromethyl,tribromomethyl), haloalkoxy (e.g. haloC₁₋₆alkoxy), hydroxy, thio (—SH),sulfonyl, sulfonamide, phenyl (which itself may be further substitutede.g., by one or more C₁₋₆alkyl, halo, hydroxy, hydroxyC₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkoxy, haloC₁₋₆alkyl,haloC₁₋₆alkoxy, cyano, nitro, OC(O)C₁₋₆alkyl, NH₂, NHC₁₋₆alkyl,NHC(O)C₁₋₆alkyl and NC₁₋₆alkylC₁₋₆alkyl), benzyl (wherein benzyl itselfmay be further substituted e.g., by one or more of C₁₋₆alkyl, halo,hydroxy, hydroxyC₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl,C₁₋₆alkoxyC₁₋₆alkoxy, haloC₁₋₆alkyl, haloC₁₋₆alkoxy, cyano, nitro,OC(O)C₁₋₆alkyl, NH₂, NHC₁₋₆alkyl, NHC(O)C₁₋₆alkyl andNC₁₋₆alkylC₁₋₆alkyl), phenoxy (wherein phenyl itself may be furthersubstituted e.g., by one or more of C₁₋₆alkyl, halo, hydroxy,hydroxyC₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkoxy,haloC₁₋₆alkyl, haloC₁₋₆alkoxy, cyano, nitro, OC(O)C₁₋₆alkyl, NH₂,NHC₁₋₆alkyl, NHC(O)C₁₋₆alkyl and NC₁₋₆alkylC₁₋₆alkyl), benzyloxy(wherein benzyl itself may be further substituted e.g., by one or moreof C₁₋₆alkyl, halo, hydroxy, hydroxyC₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyC₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkoxy, halo₁₋₆alkyl, haloC₁₋₆alkoxy,cyano, nitro, OC(O)C₁₋₆alkyl, NH₂, NHC₁₋₆alkyl, NHC(O)C₁₋₆alkyl andNC₁₋₆alkylC₁₋₆alkyl), NH₂, alkylamino (e.g. —NHC₁₋₆alkyl, such asmethylamino, ethylamino, propylamino etc), dialkylamino (e.g.—NH(C₁₋₆alkyl)₂, such as dimethylamino, diethylamino, dipropylamino),acylamino (e.g. —NHC(O)C₁₋₆alkyl, such as —NHC(O)CH₃), phenylamino (i.e.—NHphenyl, wherein phenyl itself may be further substituted e.g., by oneor more of C₁₋₆alkyl, halo, hydroxy, hydroxyC₁₋₆alkyl, hydroxyC₁₋₆alkoxyC₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkoxy, haloC₁₋₆alkyl,haloC₁₋₆alkoxy, cyano, nitro, OC(O)C₁₋₆alkyl, NH₂, NHC₁₋₆alkyl,NHC(O)C₁₋₆alkyl and NC₁₋₆alkylC₁₋₆alkyl), nitro, cyano, formyl,—C(O)-alkyl (e.g. —C(O)C₁₋₆alkyl, such as acetyl), O—C(O)-alkyl (e.g.—OC(O)C₁₋₆alkyl, such as acetyloxy), benzoyl (wherein benzyl itself maybe further substituted e.g., by one or more of C₁₋₆alkyl, halo, hydroxy,hydroxyC₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkoxy,haloC₁₋₆alkyl, haloC₁₋₆alkoxy, cyano, nitro, OC(O)C₁₋₆alkyl, NH₂,NHC₁₋₆alkyl, NHC(O)C₁₋₆alkyl and NC₁₋₆alkylC₁₋₆alkyl), benzoyloxy(wherein benzyl itself may be further substituted e.g., by one or moreof C₁₋₆alkyl, halo, hydroxy, hydroxyC₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyC₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkoxy, haloC₁₋₆alkyl,haloC₁₋₆alkoxy, cyano, nitro, OC(O)C₁₋₆alkyl, NH₂, NHC₁₋₆alkyl,NHC(O)C₁₋₆alkyl and NC₁₋₆alkylC₁₋₆alkyl), CO₂H, CO₂alkyl (e.g.CO₂C₁₋₆alkyl such as methyl ester, ethyl ester, propyl ester, butylester), CO₂phenyl (wherein phenyl itself may be further substitutede.g., by one or more of C₁₋₆alkyl, halo, hydroxy, hydroxyC₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkoxy, haloC₁₋₆alkyl,haloC₁₋₆alkoxy, cyano, nitro, OC(O)C₁₋₆alkyl, NH₂, NHC₁₋₆alkyl,NHC(O)C₁₋₆alkyl and NC₁₋₆alkylC₁₋₆alkyl), CO₂benzyl (wherein benzylitself may be further substituted e.g., by one or more of C₁₋₆alkyl,halo, hydroxy, hydroxyC₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl,C₁₋₆alkoxyC₁₋₆alkoxy, haloC₁₋₆alkyl, haloC₁₋₆alkoxy, cyano, nitro,OC(O)C₁₋₆alkyl, NH₂, NHC₁₋₆alkyl, NHC(O)C₁₋₆alkyl andNC₁₋₆alkylC₁₋₆alkyl), CONH₂, C(O)NHphenyl (wherein phenyl itself may befurther substituted e.g., by one or more of C₁₋₆alkyl, halo, hydroxy,hydroxyC₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkoxy,haloC₁₋₆alkyl, haloC₁₋₆alkoxy, cyano, nitro, OC(O)C₁₋₆alkyl, NH₂,NHC₁₋₆alkyl, NHC(O)C₁₋₆alkyl and NC₁₋₆alkylC₁₋₆alkyl), C(O)NHbenzyl(wherein benzyl itself may be further substituted e.g., by one or moreof C₁₋₆alkyl, halo, hydroxy, hydroxyC₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyC₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkoxy, haloC₁₋₆alkyl,haloC₁₋₆alkoxy, cyano, nitro, OC(O)C₁₋₆alkyl, NH₂, NHC₁₋₆alkyl,NHC(O)C₁₋₆alkyl and NC₁₋₆alkylC₁₋₆alkyl), C(O)NHalkyl (e.g. C(O)NHC₁₋₆alkyl such as methyl amide, ethyl amide, propyl amide, butyl amide)C(O)Ndialkyl (e.g. C(O)N(C₁₋₆alkyl)₂) aminoalkyl (e.g., HNC₁₋₆alkyl-,C₁₋₆alkylHN-C₁₋₆alkyl- and (C₁₋₆alkyl)₂N—C₁₋₆alkyl-), thioalkyl (e.g.,HSC₁₋₆alkyl-), carboxyalkyl (e.g., HO₂CC₁₋₆alkyl-), carboxyesteralkyl(e.g., C₁₋₆alkylO₂CC₁₋₆alkyl-), amidoalkyl (e.g., H₂N(O)CC₁₋₆alkyl-,H(C₁₋₆alkyl)N(O)CC₁₋₆alkyl-), formylalkyl (e.g., OHCC₁₋₆alkyl-),acylalkyl (e.g., C₁₋₆alkyl(O)CC₁₋₆alkyl-), nitroalkyl (e.g.,O₂NC₁₋₆alkyl-), replacement of CH₂ with C═O, replacement of CH₂ withC═S, substitution of 2 adjacent or non-adjacent carbon atoms (e.g. 1,2or 1,3) by one end each of a —O—(CH₂)_(s)—O— or —NR′—(CH₂)s-NR′— group,wherein s is 1 or 2 and each R′ is independently H or C₁₋₆alkyl, andsubstitution of 2 adjacent or non-adjacent atoms, independently selectedfrom C and N, by a C₂₋₅alkylene or C₂₋₅alkenylene group.

The term “sulfoxide”, either alone or in a compound word, refers to agroup —S(O)R wherein R is selected from hydrogen, alkyl, alkenyl,alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl.Examples of R include hydrogen, C₁₋₂₀alkyl, phenyl and benzyl.

The term “sulfonyl”, either alone or in a compound word, refers to agroup S(O)₂—R, wherein R is selected from hydrogen, alkyl, alkenyl,alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl.Examples of R include hydrogen, C₁₋₂₀alkyl, phenyl and benzyl.

The term “sulfonamide”, or “sulfonamyl” of “sulfonamido”, either aloneor in a compound word, refers to a group S(O)₂NRR wherein each R isindependently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of Rinclude hydrogen, C₁₋₂₀alkyl, phenyl and benzyl. In an embodiment atleast one R is hydrogen. In another form, both R are hydrogen.

The term “sulfamate”, either alone or in a compound word, refers to agroup —OS(O)₂NRR wherein each R is independently selected from hydrogen,alkyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl.Examples of R include hydrogen, C₁₋₂₀alkyl, phenyl and benzyl. In anembodiment at least one R is hydrogen. In another form, both R arehydrogen.

The term “sulfamide”, either alone or in a compound word, refers to agroup —NRS(O)₂NRR wherein each R is independently selected fromhydrogen, alkyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, andaralkyl. Examples of R include hydrogen, C₁₋₂₀alkyl, phenyl and benzyl.In an embodiment at least one R is hydrogen. In another form, both R arehydrogen.

A “sulfate” group refers to a group —OS(O)₂₀R wherein each R isindependently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of Rinclude hydrogen, C₁₋₂₀alkyl, phenyl and benzyl.

The term “sulfonate” refers to a group SO₃R wherein each R isindependently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of Rinclude hydrogen, C₁₋₂₀alkyl, phenyl and benzyl.

The term “thio” is intended to include groups of the formula “—SR”wherein R can be hydrogen (thiol), alkyl, alkenyl, alkynyl, aryl,carbocyclyl, heteroaryl, heterocyclyl, aralkyl, and acyl. Examples of Rinclude hydrogen, C₁₋₂₀alkyl, phenyl and benzyl.

The term, “amino” is used here in its broadest sense as understood inthe art and includes groups of the formula —NRARB wherein R_(A) andR_(B) may be independently selected from hydrogen, hydroxy alkyl,alkoxyalkyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, arylalkyl,heteroarylalkyl, carbocyclylalkyl, heterocyclylalkyl, acyl and amido,each of which may be optionally substituted as described herein. R_(A)and R_(B), together with the nitrogen to which they are attached, mayalso form a monocyclic, or fused polycyclic ring system e.g. a3-10-membered ring, particularly, 5-6 and 9-10-membered systems.Examples of “amino” include —NH₂, —NHalkyl (e.g. —NHC₁₋₂₀alkyl),—NHalkoxyalkyl, —NHaryl (e.g. —NHphenyl), —NHaralkyl (e.g. —NHbenzyl),—NHacyl (e.g. —NHC(O)C₁₋₂₀alkyl, —NHC(O)phenyl), —NHamido, (e.g.NHC(O)NHC₁₋₆alkyl, NHC(O)NH phenyl), —Ndialkyl (wherein each alkyl, forexample C₁₋₂₀, may be the same or different) and 5 or 6 membered rings,optionally containing one or more same or different heteroatoms (e.g. O,N and S). Reference to groups written as “[group]amino” is intended toreflect the nature of the R_(A) and R_(B) groups. For example,“alkylamino” refers to —NRARB where one of R_(A) or R_(B) is alkyl.“Dialkylamino” refers to —NRARB where R_(A) and R_(B) are each(independently) an alkyl group.

The term “amido” is used here in its broadest sense as understood in theart and includes groups having the formula C(O)NRARB, wherein R_(A) andR_(B) are as defined as above. Examples of amido include C(O)NH₂,C(O)NHalkyl (e.g. C₁₋₂₀alkyl), C(O)NHaryl (e.g. C(O)NHphenyl),C(O)NHaralkyl (e.g. C(O)NHbenzyl), C(O)NHacyl (e.g.C(O)NHC(O)C₁₋₂₀alkyl, C(O)NHC(O)phenyl), C(O)Nalkylalkyl (wherein eachalkyl, for example C₁₋₂₀, may be the same or different) and 5 or 6membered rings, optionally containing one or more same or differentheteroatoms (e.g. O, N and S).

The term “carboxy ester” is used here in its broadest sense asunderstood in the art and includes groups having the formula —CO₂R,wherein R may be selected from groups including alkyl, alkenyl, alkynyl,aryl, carbocyclyl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl,carbocyclylalkyl, heterocyclylalkyl, aralkenyl, heteroarylalkenyl,carbocyclylalkenyl, heterocyclylalkenyl, aralkynyl, heteroarylalkynyl,carbocyclylalkynyl, heterocyclylalkynyl, and acyl, each of which may beoptionally substituted. Some examples of carboxy ester include—CO₂C₁₋₂₀alkyl, —CO₂aryl (e.g. —CO₂phenyl), —CO₂arC₁₋₂₀alkyl (e.g. —CO₂benzyl).

The term “phosphonate” refers to a group —P(O)(OR₂) wherein R isindependently selected from hydrogen, alkyl, aryl, heteroaryl,heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of R includehydrogen, C₁₋₂₀alkyl, phenyl and benzyl.

The term “phosphate” refers to a group —OP(O)(OR)₂ wherein R isindependently selected from hydrogen, alkyl, aryl, heteroaryl,heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of R includehydrogen, C₁₋₂₀alkyl, phenyl and benzyl.

Carboxyclic isosteres are groups which can exhibit the same or similarproperties as a carboxylic group. Some examples of carboxylic acidisosteres include: —SO₃H, —SO₂NHR, —PO₂R₂, —CN, —PO₂R₂, —OH, —OR, —SH,—SR, —NHCOR, —NR₂, —CONR₂, —CONH(O)R, —CONHNHSO₂R, —COHNSO₂R and—CONR—CN, where R is selected from H, alkyl (such as C₁₋₆ alkyl), phenyland benzyl. Other carboxylic acid isosteres include carbocyclic andheterocyclic groups such as:

As used herein, reference to IRAP inhibitor or inhibitor of IRAP alsoincludes a pharmaceutically acceptable salt, solvate, polymorph orprodrug thereof.

The term ‘pharmaceutically-acceptable salts’ refers to those saltswhich, within the scope of sound medical judgement, are suitable for usein contact with the tissues of humans and animals without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. S. M. Berge et al. describepharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 1977, 66:1-19. The salts include relatively non-toxic,inorganic and organic acid salts of any small molecule inhibitors, asappropriate.

Examples of such inorganic acids are hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid. Appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,heterocyclic carboxylic and sulfonic classes of organic acids, examplesof which are formic, acetic, propionic, succinic, glycolic, gluconic,lactic, malic, tartaric, citric, ascorbic, glucoronic, fumaric, maleic,pyruvic, alkyl sulfonic, arylsulfonic, aspartic, glutamic, benzoic,anthranilic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic,mandelic, ambonic, pamoic, pantothenic, sulfanilic,cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, galactaric,and galacturonic acids. Suitable pharmaceutically acceptable baseaddition salts of the compounds of the present invention includemetallic salts made from lithium, sodium, potassium, magnesium, calcium,aluminium, and zinc, and organic salts made from organic bases such ascholine, diethanolamine, morpholine. Alternatively, organic salts madefrom N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N methylglucamine),procaine, ammonium salts, quaternary salts such as tetramethylammoniumsalt, amino acid addition salts such as salts with glycine and arginine.

For example, alkali metal salts (K, Na) and alkaline earth metal salts(Ca, Mg) may be used if deemed appropriate for the structure, but againany pharmaceutically acceptable, non-toxic salt may be used whereappropriate. The Na- and Ca-salts are preferred.

Pharmaceutically acceptable solvates, including hydrates, of suchcompounds and such salts are also intended to be included within thescope of this invention.

In the case of small molecule inhibitors that are solids, it will beunderstood by those skilled in the art that the compounds, agents andsalts may exist in different crystalline or polymorphic forms, all ofwhich are intended to be within the scope of the present invention andspecified formulae.

The term ‘polymorph’ includes any crystalline form of compounds of anycompound described herein, such as anhydrous forms, hydrous forms,solvate forms and mixed solvate forms.

An antibody inhibitor of IRAP can be produced via techniques known inthe art to generate an antibody against IRAP and then those antibodiescan be screened for IRAP inhibitory activity using assays as describedherein. For example, monoclonal antibodies can be prepared as follows.Immunization of mice or other appropriate host animal by an IRAP offragment thereof. Immunization with IRAP of fragment thereof and/oradjuvant may be by multi-point injection usually subcutaneous injectionor intraperitoneal injection. IRAP of fragment thereof may be conjugatedto a carrier, such as serum albumin, or soybean trypsin on inhibitor, anantigen to enhance immunogenicity in the host. The preferred animalsystem for generating hybridomas is the murine system. Immunizationprotocols and techniques for isolation of immunized splenocytes forfusion are well known in the art. Fusion cell partners (e.g., murinemyeloma cell lines SP2/0, NS0, NS1, rat myeloma Y3, rabbit myeloma 240E1, human K6H6), fusion and screening procedures are also well known inthe art (Galfre et al., 1977; Gefter et al., 1977; Galfre et al., 1979;Dangl et al., 1982; Spieker-Polet et al., 1995).

The phrase ‘therapeutically effective amount’ generally refers to anamount of one or more inhibitors, or, if a small molecule inhibitor, apharmaceutically acceptable salt, polymorph or prodrug thereof of thepresent invention that (i) treats the particular disease, condition, ordisorder, (ii) attenuates, ameliorates, or eliminates one or moresymptoms of the particular disease, condition, or disorder, or (iii)delays the onset of one or more symptoms of the particular disease,condition, or disorder described herein.

“Fibrosis”, “Fibrotic disease” or “Fibro proliferative disease” meansthe formation of excess fibrous connective tissue in a reparativeprocess upon injury. Scarring is a result of continuous fibrosis thatobliterates the affected organs or tissues architecture. As a result ofabnormal reparative processes, which do not clear the formed scartissue, fibrosis progresses further. Fibrosis can be found in varioustissues, including the heart, the lungs, the liver, the skin, bloodvessels and the kidneys. Examples of fibrosis are described herein andinclude pulmonary fibrosis, liver cirrhosis, systemic sclerosis,progressive kidney disease and cardiac fibrosis associated with variouscardiovascular diseases.

An individual may be identified as having fibrosis by determining if asubject has organ dysfunction, scarring, alteration of normalextracellular matrix balance, increase in collagen deposition, increasedcollagen volume fraction, differentiation of fibroblasts tomyofibroblasts, reduction in the level of matrix metalloproteinases andincrease in the level of tissue Inhibitors of matrix metalloproteinases,increased levels of either N-terminal or C-terminal propeptide of type Iprocollagen (PINP or PICP) and decreased levels of C-terminaltelopeptide of Type I Collagen (CTP or CITP), increased collagendeposition and impaired cardiac function measured by various noninvasiveimaging techniques, impaired renal function measured by increasedproteinurea and albuminurea, decreased glomerular filtration rate,doubling of plasma creatinine levels.

Preferably the fibrotic disease is associated upregulation of IRAPexpression and/or activity. IRAP expression or activity can be measuredby any assay described herein.

Organ fibrosis related to tissue injury includes fibrosis associatedwith cardiovascular disease and fibrosis that has occurred following anorgan transplant, such as a kidney or liver transplant.

According to a preferred embodiment of the invention, the pulmonaryfibrosis is idiopathic pulmonary fibrosis, sarcoidosis, cystic fibrosis,familial pulmonary fibrosis, silicosis, asbestosis, coal worker'spneumoconiosis, carbon pneumoconiosis, hypersensitivity pneumonitides,pulmonary fibrosis caused by inhalation of inorganic dust, pulmonaryfibrosis caused by an infectious agent, pulmonary fibrosis caused byinhalation of noxious gases, aerosols, chemical dusts, fumes or vapours,drug-induced interstitial lung disease, or pulmonary hypertension.

According to a preferred embodiment of the invention, the liver fibrosisis resulting from a chronic liver disease, hepatitis B virus infection,hepatitis C virus infection, hepatitis D virus infection,schistosomiasis, alcoholic liver disease or non-alcoholicsteatohepatitis, non-alcoholic fatty liver disease, obesity, diabetes,protein malnutrition, coronary artery disease, auto-immune hepatitis,cystic fibrosis, alpha-1-antitrypsin deficiency, primary biliarycirrhosis, drug reaction and exposure to toxins.

According to a preferred embodiment of the invention, the skin fibrosisis scarring, hypertrophic scarring, keloid scarring, dermal fibroticdisorder, psoriasis or scleroderma. Said scarring may derived from aburn, a trauma, a surgical injury, a radiation or an ulcer. Said ulcercan be a diabetic foot ulcer, a venous leg ulcer or a pressure ulcer.

As used herein, “preventing” or “prevention” is intended to refer to atleast the reduction of likelihood of the risk of (or susceptibility to)acquiring a disease or disorder (i.e., causing at least one of theclinical symptoms of the disease not to develop in a patient that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease). Biological and physiologicalparameters for identifying such patients are provided herein and arealso well known by physicians. For example, prevention of age-inducedcardiac fibrosis, or cardiac or renal fibrosis associated withhypertensive heart disease, hypertensive cardiomyopathy or heartfailure, or nephropathy with or without associated diabetes, may becharacterised by an absence of interstitial collagen deposition, or anabsence of an increase in interstitial collagen deposition if collagendeposition is already detectable in a subject.

The terms “treatment” or “treating” of a subject includes theapplication or administration of a compound of the invention to asubject (or application or administration of a compound of the inventionto a cell or tissue from a subject) with the purpose of delaying,slowing, stabilizing, curing, healing, alleviating, relieving, altering,remedying, less worsening, ameliorating, improving, or affecting thedisease or condition, the symptom of the disease or condition, or therisk of (or susceptibility to) the disease or condition. The term“treating” refers to any indication of success in the treatment oramelioration of an injury, pathology or condition, including anyobjective or subjective parameter such as abatement; remission;lessening of the rate of worsening; lessening severity of the disease;stabilization, diminishing of symptoms or making the injury, pathologyor condition more tolerable to the subject; slowing in the rate ofdegeneration or decline; making the final point of degeneration lessdebilitating; or improving a subject's physical or mental well-being.

The existence of, improvement in, treatment of or prevention of afibrotic disease may be by any clinically or biochemically relevantmethod of the subject or a biopsy therefrom. For example, a parametermeasured may be the presence of fibrosis, the content of collagen,fibronectin, or another extracellular matrix protein, the phosphatidicacid level or choline level, the proliferation rate of the cells or anyextracellular matrix components in the cells or transdifferentiation ofthe cells to myofibroblasts. For example, inhibition of kidney fibrosiscan be detected by preventing a further loss of kidney function asmeasured by albuminurea or proteinurea, increased serum creatinine, areduction in active fibrosis as measured by reduced levels of collagenfragments in urine samples, and by a reduction in the presence ofmyofibroblasts on kidney biopsy tissue. Further, for example, in lungfibrosis, a positive response to therapy would be to prevent a furtherdecline in lung function as measured by spirometry, bodyplethysmography, and lung diffusion capacity. In addition, blood levelsof collagen fragments would also be reduced.

Reversing fibrosis as described herein includes inhibiting synthesisand/or enhancing degradation of collagen. A clinically or biochemicallyobservable consequence of a reversal of fibrosis is a reduction infibrotic tissue formed as a response to ageing or tissue injury.Reversing fibrosis also may include a clinically or biochemicallyobservable reduction in any characteristic or symptom of fibrosis asdescribed herein at a time after treatment has commenced compared to atime prior to treatment commencing.

The term “antagonizing” used herein is intended to mean “decreasing” or“reducing”. A sufficient period of time can be during one week, orbetween 1 week to 1 month, or between 1 to 2 months, or 2 months ormore. For chronic condition, the compound of the present invention canbe advantageously administered for life time period.

The term “pulmonary fibrosis” or “lung fibrosis” means the formation ordevelopment of excess fibrous connective tissue (fibrosis) in the lungthereby resulting in the development of scarred (fibrotic) tissue. Moreprecisely, pulmonary fibrosis is a chronic disease that causes swellingand scarring of the alveoli and interstitial tissues of the lungs. Thescar tissue replaces healthy tissue and causes inflammation. Thischronic inflammation is, in turn, the prelude to fibrosis. This damageto the lung tissue causes stiffness of the lungs which subsequentlymakes breathing more and more difficult.

The term “liver fibrosis” means the formation or development of excessfibrous connective tissue (fibrosis) in the liver thereby resulting inthe development of scarred (fibrotic) tissue. The scarred tissuereplaces healthy tissue by the process of fibrosis and leads tosubsequent cirrhosis of the liver.

The term “skin fibrosis” or “dermal fibrosis” means the excessiveproliferation of epithelial cells or fibrous connective tissue(fibrosis) thereby resulting in the development of scarred (fibrotic)tissue. The scarred tissue replaces healthy tissue by the process offibrosis and may be the prelude of systemic scleroderma. Skin fibrosisis intended to cover the fibrosis of any skin tissue and epithelialcells including, without limitation, blood vessels and veins, internalcavity of an organ or a gland such as ducts of submandibular,gallbladder, thyroid follicles, sweat gland ducts, ovaries, kidney;epithelial cells of gingival, tongue, palate, nose, larynx, oesophagus,stomach, intestine, rectum, anus and vagina; derma, scar, skin andscalp. The compounds of the present invention may be active forpromoting healing of wound and one or more of the following activities:

-   -   improving collagen organization and/or reducing wound        cellularity in said wound;    -   reducing collagen overproduction by fibroblast and epithelial        cells in said wound;    -   reducing epithelial mesenchymal transition in said wound;    -   reducing fibroblast migration and activation in said wound;    -   reducing and/or inhibiting dermal thickening in said wound;    -   reducing and/or inhibiting recruitment of inflammatory cells to        said wound.

The term “cardiac fibrosis” or “heart fibrosis” means an abnormalthickening of the heart valves due to inappropriate proliferation ofcardiac fibroblasts but more commonly refers to the proliferation offibroblasts in the cardiac muscle. Fibrocyte cells normally secretecollagen, and function to provide structural support for the heart. Whenover-activated this process causes thickening and fibrosis of the valvesand heart muscle itself, with white tissue building up primarily on thetricuspid or mitral valve, but also occurring on the pulmonary or aorticvalve. The thickening and loss of flexibility eventually may lead tovalvular dysfunction and right-sided or left-sided heart failure. Ingeneral, prophylactic and therapeutic uses comprise the administrationof a compound as described herein to a subject, preferably a humanpatient in need thereof.

“Idiopathic pulmonary fibrosis (IPF)” is a specific manifestation ofidiopathic interstitial pneumonia (lIIP), a type of interstitial lungdisease. Interstitial lung disease, also known as diffuse parenchymallung disease (DPLD), refers to a group of lung diseases affecting theinterstitium. Microscopically, lung tissue from IPF patients shows acharacteristic set of histological features known as usual interstitialpneumonia (UIP). UIP is therefore the pathologic presentation of IPF.

Exemplary forms of fibrosis include, but are not limited to, cardiacfibrosis, liver fibrosis, kidney fibrosis, lung fibrosis, vascularfibrosis, dermal scarring and keloids, and Alzheimer's disease. In stillfurther embodiments, cardiac fibrosis is associated with hypertension,hypertensive heart disease (HHD), hypertensive cardiomyopathy (HCM),myocardial infarction (MI), and restenosis or as a result of impairedrenal function resulting from renal fibrosis.

Preferably, the fibrosis is kidney fibrosis. The kidney fibrosis mayinclude, but not be limited to, diabetic nephropathy, vesicoureteralreflux, tubulointerstitial renal fibrosis, glomerulonephritis orglomerular nephritis (GN), focal segmental glomerulosclerosis,membranous glomerulonephritis, or mesangiocapillary GN. The liverfibrosis may include, but not be limited to, cirrhosis, and associatedconditions such as chronic viral hepatitis, non-alcoholic fatty liverdisease (NAFLD), alcoholic steatohepatitis (ASH), non-alcoholicsteatohepatitis (NASH), primary biliary cirrhosis (PBC), biliarycirrhosis, autoimmune hepatitis). Lung fibrosis may include idiopathicpulmonary fibrosis (IPF) or cryptogenic fibrosing alveolitis, chronicfibrosing interstitial pneumonia, interstitial lung disease (ILD), anddiffuse parenchymal lung disease (DPLD)). Cardiac fibrosis, congestiveheart failure, cardiomyopathy, post-myocardial infarction defects inheart function; peripheral vascular disease; rheumatoid arthritis;glaucoma; age-related macular degeneration (wet AMD and dry AMD);emphysema, chronic obstructive pulmonary disease (COPD); multiplesclerosis; and chronic asthma may also be prevented, treated, orameliorated with compositions, methods or uses as described herein.

As a result of any method or use as described herein, inhibition of IRAPmay improve heart function and decrease infarct area followingischemic-reperfusion (I/R) injury.

In a preferred form, the fibrotic disease is cardiac, renal, liver orinterstitial fibrosis.

Scleroderma (systemic sclerosis), a chronic systemic autoimmune diseasecharacterised by hardening (sclero) of the skin (derma) and internalorgans (in severe cases). Clinically, patient stratification and drugefficacy can be measured through biopsy/visualization of reduced skinlesions and other objective measures assessed over 24 and 48 weeks. Assuch, diabetic nephropathy, IgA nephropathy or scleroderma are alsofibrotic conditions for treatment and/or prevention.

In the cardiovascular system a progressive age-related deposition ofcollagen in the vascular wall and in the cardiac interstitial andperivascular space, or collagen deposition related to cardiovascular orrenal disease, leads to reduction of myocardial and arterial compliance.

The frequency of administration may be once daily, or 2 or 3 time daily.The treatment period may be for the duration of the detectable disease.

Typically, a therapeutically effective dosage is formulated to contain aconcentration (by weight) of at least about 0.1% up to about 50% ormore, and all combinations and sub-combinations of ranges therein. Thecompositions can be formulated to contain one or more compoundsaccording to Formula I, or a pharmaceutically acceptable salt, polymorphor prodrug thereof in a concentration of from about 0.1 to less thanabout 50%, for example, about 49, 48, 47, 46, 45, 44, 43, 42, 41 or 40%,with concentrations of from greater than about 0.1%, for example, about0.2, 0.3, 0.4 or 0.5%, to less than about 40%, for example, about 39,38, 37, 36, 35, 34, 33, 32, 31 or 30%. Exemplary compositions maycontain from about 0.5% to less than about 30%, for example, about 29,28, 27, 26, 25, 25, 24, 23, 22, 21 or 20%, with concentrations of fromgreater than about 0.5%, for example, about 0.6, 0.7, 0.8, 0.9 or 1%, toless than about 20%, for example, about 19, 18, 17, 16, 15, 14, 13, 12,11 or 10%. The compositions can contain from greater than about 1% forexample, about 2%, to less than about 10%, for example about 9 or 8%,including concentrations of greater than about 2%, for example, about 3or 4%, to less than about 8%, for example, about 7 or 6%. The activeagent can, for example, be present in a concentration of about 5%. Inall cases, amounts may be adjusted to compensate for differences inamounts of active ingredients actually delivered to the treated cells ortissue.

Although the invention finds application in humans, the invention isalso useful for therapeutic veterinary purposes. The invention is usefulfor domestic or farm animals such as cattle, sheep, horses and poultry;for companion animals such as cats and dogs; and for zoo animals.

Pharmaceutical compositions may be formulated for any appropriate routeof administration including, for example, topical (for example,transdermal or ocular), oral, buccal, nasal, vaginal, rectal orparenteral administration. The term parenteral as used herein includessubcutaneous, intradermal, intravascular (for example, intravenous),intramuscular, spinal, intracranial, intrathecal, intraocular,periocular, intraorbital, intrasynovial and intraperitoneal injection,as well as any similar injection or infusion technique. In certainembodiments, compositions in a form suitable for oral use or parenteraluse are preferred. Suitable oral forms include, for example, tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, or syrups or elixirs. Withinyet other embodiments, compositions provided herein may be formulated asa lyophilizate.

The various dosage units are each preferably provided as a discretedosage tablet, capsules, lozenge, dragee, gum, or other type of solidformulation. Capsules may encapsulate a powder, liquid, or gel. Thesolid formulation may be swallowed, or may be of a suckable or chewabletype (either frangible or gum-like). The present invention contemplatesdosage unit retaining devices other than blister packs; for example,packages such as bottles, tubes, canisters, packets. The dosage unitsmay further include conventional excipients well-known in pharmaceuticalformulation practice, such as binding agents, gellants, fillers,tableting lubricants, disintegrants, surfactants, and colorants; and forsuckable or chewable formulations.

Compositions intended for oral use may further comprise one or morecomponents such as sweetening agents, flavouring agents, colouringagents and/or preserving agents in order to provide appealing andpalatable preparations. Tablets contain the active ingredient inadmixture with physiologically acceptable excipients that are suitablefor the manufacture of tablets. Such excipients include, for example,inert diluents such as calcium carbonate, sodium carbonate, lactose,calcium phosphate or sodium phosphate, granulating and disintegratingagents such as corn starch or alginic acid, binding agents such asstarch, gelatine or acacia, and lubricating agents such as magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatinecapsules wherein the active ingredient is mixed with an inert soliddiluent such as calcium carbonate, calcium phosphate or kaolin, or assoft gelatine capsules wherein the active ingredient is mixed with wateror an oil medium such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active ingredient(s) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include suspending agents such as sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as naturally-occurringphosphatides (for example, lecithin), condensation products of analkylene oxide with fatty acids such as polyoxyethylene stearate,condensation products of ethylene oxide with long chain aliphaticalcohols such as heptadecaethyleneoxycetanol, condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol mono-oleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides such as polyethylene sorbitan monooleate. Aqueoussuspensions may also comprise one or more preservatives, for exampleethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, oneor more flavouring agents, and one or more sweetening agents, such assucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oily suspensionsmay contain a thickening agent such as beeswax, hard paraffin or cetylalcohol. Sweetening agents such as those set forth above, and/orflavouring agents may be added to provide palatable oral preparations.Such suspensions may be preserved by the addition of an antioxidant suchas ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, such as sweetening, flavouring and colouringagents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil such as olive oil orarachis oil, a mineral oil such as liquid paraffin, or a mixturethereof. Suitable emulsifying agents include naturally-occurring gumssuch as gum acacia or gum tragacanth, naturally-occurring phosphatidessuch as soy bean lecithin, and esters or partial esters derived fromfatty acids and hexitol, anhydrides such as sorbitan monoleate, andcondensation products of partial esters derived from fatty acids andhexitol with ethylene oxide such as polyoxyethylene sorbitan monoleate.An emulsion may also comprise one or more sweetening and/or flavouringagents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso comprise one or more demulcents, preservatives, flavouring agentsand/or colouring agents.

Compounds may be formulated for local or topical administration, such asfor topical application to the skin. Formulations for topicaladministration typically comprise a topical vehicle combined with activeagent(s), with or without additional optional components.

For any of the fibrotic diseases described herein, when the compound ofthe present invention is topically administered to a human, thetherapeutically effective amount of a compound corresponds to preferablybetween about 0.01 to about 10% (w/w), or between about 0.1 to 10%(w/w), or between about 1.0 to about 10% (w/w), between about 0.1 toabout 5% (w/w), or between about 1.0 to about 5% (w/w). In any offibrotic diseases described herein, when the compound of the presentinvention is orally administered to a subject, the therapeuticallyeffective amount of a compound corresponds preferably between about 1 toabout 50 mg/kg, or between about 1 to 35 mg/kg. or between about 1 to 25mg/kg, or between about 1 to about 10 mg/kg, between about 5 to about 25mg/kg, or between about 10 to about 20 mg/kg.

‘Prodrug’ means a compound which is convertible in vivo by metabolicmeans (e.g. by hydrolysis, reduction or oxidation) to a compound of thepresent invention. For example an ester prodrug of a compound of thepresent invention containing a hydroxyl group may be convertible byhydrolysis in vivo to the parent molecule. Where esters can be formed,suitable esters are, for example, acetates, citrates, lactates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoates, gestisates,isethionates, di-p-toluoyltartrates, methanesulphonates,ethanesulphonates, benzenesulphonates, p-toluenesulphonates,cyclohexylsulphamates and quinates.

Prodrugs prepared through common variations to the structure of one ormore compounds according to Formula I, II or Ill, or a pharmaceuticallyacceptable salt, polymorph or prodrug thereof will be well-known to aperson skilled in the art and are included herein. For example, thetypes of prodrugs described in Zawilska, J. B. et al. PharmacologicalReports, 2013, 65, 1-14 are encompassed in this application where theyare relevant to relevant compound's structure and route ofadministration.

Suitable topical vehicles and additional components are well known inthe art, and it will be apparent that the choice of a vehicle willdepend on the particular physical form and mode of delivery. Topicalvehicles include organic solvents such as alcohols (for example,ethanol, iso-propyl alcohol or glycerine), glycols such as butylene,isoprene or propylene glycol, aliphatic alcohols such as lanolin,mixtures of water and organic solvents and mixtures of organic solventssuch as alcohol and glycerine, lipid-based materials such as fattyacids, acylglycerols including oils such as mineral oil, and fats ofnatural or synthetic origin, phosphoglycerides, sphingolipids and waxes,protein-based materials such as collagen and gelatine, silicone-basedmaterials (both nonvolatile and volatile), and hydrocarbon-basedmaterials such as microsponges and polymer matrices.

A composition may further include one or more components adapted toimprove the stability or effectiveness of the applied formulation, suchas stabilizing agents, suspending agents, emulsifying agents, viscosityadjusters, gelling agents, preservatives, antioxidants, skin penetrationenhancers, moisturizers and sustained release materials. Examples ofsuch components are described in Martindale—The Extra Pharmacopoeia(Pharmaceutical Press, London 1993) and Martin (ed.), Remington'sPharmaceutical Sciences. Formulations may comprise microcapsules, suchas hydroxymethylcellulose or gelatine-microcapsules, liposomes, albuminmicrospheres, microemulsions, nanoparticles or nanocapsules.

A topical formulation may be prepared in a variety of physical formsincluding, for example, solids, pastes, creams, foams, lotions, gels,powders, aqueous liquids, emulsions, sprays and skin patches. Thephysical appearance and viscosity of such forms can be governed by thepresence and amount of emulsifier(s) and viscosity adjuster(s) presentin the formulation. Solids are generally firm and non-pourable andcommonly are formulated as bars or sticks, or in particulate form.Solids can be opaque or transparent, and optionally can containsolvents, emulsifiers, moisturizers, emollients, fragrances,dyes/colorants, preservatives and other active ingredients that increaseor enhance the efficacy of the final product. Creams and lotions areoften similar to one another, differing mainly in their viscosity. Bothlotions and creams may be opaque, translucent or clear and often containemulsifiers, solvents, and viscosity adjusting agents, as well asmoisturizers, emollients, fragrances, dyes/colorants, preservatives andother active ingredients that increase or enhance the efficacy of thefinal product. Gels can be prepared with a range of viscosities, fromthick or high viscosity to thin or low viscosity. These formulations,like those of lotions and creams, may also contain solvents,emulsifiers, moisturizers, emollients, fragrances, dyes/colorants,preservatives and other active ingredients that increase or enhance theefficacy of the final product. Liquids are thinner than creams, lotions,or gels, and often do not contain emulsifiers. Liquid topical productsoften contain solvents, emulsifiers, moisturizers, emollients,fragrances, dyes/colorants, preservatives and other active ingredientsthat increase or enhance the efficacy of the final product.

Emulsifiers for use in topical formulations include, but are not limitedto, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers likepolyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12,ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate andglyceryl stearate. Suitable viscosity adjusting agents include, but arenot limited to, protective colloids or nonionic gums such ashydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silica,microcrystalline wax, beeswax, paraffin, and cetyl palmitate. A gelcomposition may be formed by the addition of a gelling agent such aschitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol,polyquaterniums, hydroxyethylceilulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate.Suitable surfactants include, but are not limited to, nonionic,amphoteric, ionic and anionic surfactants. For example, one or more ofdimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60,polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleylbetaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammoniumlaureth sulfate may be used within topical formulations.

Preservatives include, but are not limited to, antimicrobials such asmethylparaben, propylparaben, sorbic acid, benzoic acid, andformaldehyde, as well as physical stabilizers and antioxidants such asvitamin E, sodium ascorbate/ascorbic acid and propyl gallate. Suitablemoisturizers include, but are not limited to, lactic acid and otherhydroxy acids and their salts, glycerine, propylene glycol, and butyleneglycol. Suitable emollients include lanolin alcohol, lanolin, lanolinderivatives, cholesterol, petrolatum, isostearyl neopentanoate andmineral oils. Suitable fragrances and colours include, but are notlimited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitableadditional ingredients that may be included in a topical formulationinclude, but are not limited to, abrasives, absorbents, anticakingagents, antifoaming agents, antistatic agents, astringents (such aswitch hazel), alcohol and herbal extracts such as chamomile extract,binders/excipients, buffering agents, chelating agents, film formingagents, conditioning agents, propellants, opacifying agents, pHadjusters and protectants.

Typical modes of delivery for topical compositions include applicationusing the fingers, application using a physical applicator such as acloth, tissue, swab, stick or brush, spraying including mist, aerosol orfoam spraying, dropper application, sprinkling, soaking, and rinsing.Controlled release vehicles can also be used, and compositions may beformulated for transdermal administration (for example, as a transdermalpatch).

A pharmaceutical composition may be formulated as inhaled formulations,including sprays, mists, or aerosols. This may be particularly preferredfor treatment of pulmonary fibrosis. For inhalation formulations, thecomposition or combination provided herein may be delivered via anyinhalation methods known to a person skilled in the art. Such inhalationmethods and devices include, but are not limited to, metered doseinhalers with propellants such as CFC or HFA or propellants that arephysiologically and environmentally acceptable. Other suitable devicesare breath operated inhalers, multidose dry powder inhalers and aerosolnebulizers. Aerosol formulations for use in the subject method typicallyinclude propellants, surfactants and co-solvents and may be filled intoconventional aerosol containers that are closed by a suitable meteringvalve.

Inhalant compositions may comprise liquid or powdered compositionscontaining the active ingredient that are suitable for nebulization andintrabronchial use, or aerosol compositions administered via an aerosolunit dispensing metered doses. Suitable liquid compositions comprise theactive ingredient in an aqueous, pharmaceutically acceptable inhalantsolvent such as isotonic saline or bacteriostatic water. The solutionsare administered by means of a pump or squeeze-actuated nebulized spraydispenser, or by any other conventional means for causing or enablingthe requisite dosage amount of the liquid composition to be inhaled intothe patient's lungs. Suitable formulations, wherein the carrier is aliquid, for administration, as for example, a nasal spray or as nasaldrops, include aqueous or oily solutions of the active ingredient.

Pharmaceutical compositions may also be prepared in the form ofsuppositories such as for rectal administration. Such compositions canbe prepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Suitable excipients include, for example, cocoa butter and polyethyleneglycols.

Pharmaceutical compositions may be formulated as sustained releaseformulations such as a capsule that creates a slow release of modulatorfollowing administration. Such formulations may generally be preparedusing well-known technology and administered by, for example, oral,rectal or subcutaneous implantation, or by implantation at the desiredtarget site. Carriers for use within such formulations arebiocompatible, and may also be biodegradable. Preferably, theformulation provides a relatively constant level of modulator release.The amount of modulator contained within a sustained release formulationdepends upon, for example, the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

In another embodiment there is provided a kit or article of manufactureincluding one or more inhibitors of IRAP as described herein, or apharmaceutically acceptable salt, polymorph or prodrug thereof and/orpharmaceutical composition as described above.

In other embodiments there is provided a kit for use in a therapeutic orprophylactic application mentioned above, the kit including:

-   -   a container holding a therapeutic composition in the form of one        or more inhibitors of IRAP as described herein, or a        pharmaceutically acceptable salt, polymorph or prodrug thereof        or pharmaceutical composition;    -   a label or package insert with instructions for use.

In certain embodiments the kit may contain one or more further activeprinciples or ingredients for treatment of a fibrotic disease.

The kit or “article of manufacture” may comprise a container and a labelor package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, blister pack,etc. The containers may be formed from a variety of materials such asglass or plastic. The container holds a therapeutic composition which iseffective for treating the condition and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). The labelor package insert indicates that the therapeutic composition is used fortreating the condition of choice. In one embodiment, the label orpackage insert includes instructions for use and indicates that thetherapeutic or prophylactic composition can be used to treat a fibroticdisease described herein.

The kit may comprise (a) a therapeutic or prophylactic composition; and(b) a second container with a second active principle or ingredientcontained therein. The kit in this embodiment of the invention mayfurther comprise a package insert indicating the composition and otheractive principle can be used to treat a disorder or prevent acomplication stemming from a fibrotic disease described herein.Alternatively, or additionally, the kit may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

In certain embodiments the therapeutic composition may be provided inthe form of a device, disposable or reusable, including a receptacle forholding the therapeutic, prophylactic or pharmaceutical composition. Inone embodiment, the device is a syringe. The device may hold 1-2 mL ofthe therapeutic composition. The therapeutic or prophylactic compositionmay be provided in the device in a state that is ready for use or in astate requiring mixing or addition of further components.

It will be understood, that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, and rate ofexcretion, drug combination (i.e. other drugs being used to treat thepatient), and the severity of the particular disorder undergoingtherapy.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

It will be understood that these examples are intended to demonstratethese and other aspects of the invention and although the examplesdescribe certain embodiments of the invention, it will be understoodthat the examples do not limit these embodiments to these things.Various changes can be made and equivalents can be substituted andmodifications made without departing from the aspects and/or principlesof the invention mentioned above. All such changes, equivalents andmodifications are intended to be within the scope of the claims setforth herein.

EXAMPLES

Generation of the IRAP Knockout Mice Global IRAP deficient (IRAP^(−/−))mice were generated by Ozgene Pty Ltd, (Perth, Australia) as previouslydescribed (Albiston, 2009). Offspring were genotyped by PCR using theoligonucleotides GATAAGATAGTAGGGGAGA (SEQ ID NO: 7),CAATAGAGGTACAGTCACCA (SEQ ID NO: 8) and GGAGAATAAGGGCTGTGAGAGA (SEQ IDNO: 9) (Genetic accession NT______039643) with resultant wildtype allelePCR product of 384 bp and knockout allele of 1041 bp. C₅₇BL/6J mice wereused as wild-type (WT) controls. Young mice aged between 4-6 months oldand aged mice of 18-22 months old of both strains weighing between 35-50g were obtained from Monash Animal Research Laboratories (ARL). Micewere fed a normal diet ad libitum and housed in the Pharmacology AnimalHouse, Monash University in standard mouse cages (approximately 4 miceper cage) at 21±1-5° C., with a 12 hour light/dark room. All treatmentsand experimental procedures were approved by the Monash UniversityAnimal Ethics Committee (Ethics # SOBSB/PHAR/2010/23).

Drug Treatment and Surgical Procedures

There are 8 different sets of in vivo experiments in this study:

A) Phenotypic characterisation of the heart and blood vessels in globalIRAP knockout mice and their WT controls treated for 4 weeks withAngiotensin (Ang) II (800 ng/kg/min; s.c.) where mouse hearts and bloodvessels were compared to tissue obtained from young WT and IRAP^(−/−)mice treated with saline.

B) Prevention of Ang II-induced changes in the cardiovascular systemfollowing IRAP inhibitor treatment. In the prevention model, WT micewere treated with Ang II (800 ng/kg/min; s.c.)±the IRAP inhibitor(HFI-419; 500 ng/kg/min for 28 days) or HFI-vehicle (1 DMSO:3 HBC).

C) Phenotypic characterisation of the aged heart, kidney and bloodvessels in the global IRAP knockout mice where aged WT and IRAP^(−/−)mouse hearts, kidneys and blood vessels were compared to tissue obtainedfrom young WT and IRAP^(−/−) mice.

D) Reversal of the age-induced changes in the cardiovascular systemfollowing IRAP inhibitor treatment. In the reversal model, aged WT micewere treated with either saline, IRAP inhibitor (HFI-419 at 500ng/kg/min; compound 1 at 500 ng/kg/min; compound 2 at 50 ng/kg/min) orHFI-vehicle (1 DMSO: 3 HBC) for 4 weeks.

E) Prevention of ischemic-reperfusion injury in isolated hearts takenfrom aged global IRAP knockout mice and aged IRAP inhibitor (HFI-419 at500 ng/kg/min; s.c.) treated WT mice compared to age-matchedvehicle-treated (1 DMSO:3 HBC; s.c.) WT controls.

F) Phenotypic characterization of cardiac function usingechocardiography in the aged global IRAP knockout mice compared to agedand young WT mice.

G) Phenotypic characterization of liver steatosis in IRAP knockout micein a high fat diet (HFD) model.

H) Reversal of the salt-induced fibrosis in the liver following IRAPinhibitor treatment.

All mice which underwent surgery were anaesthetized with Isoflurane(Isorrane) (5% induction and 2.5% maintenance) and an incision made inthe midscapular region through which osmotic minipumps (Alzet model2004, Alza Corp) were inserted for subcutaneous drug administration. Theincision area was sutured with 6/0 DY silk (Dynek Pty Ltd) andantibiotic powder applied (Cicatrin, Pfizer) followed by intramuscularinjection of the analgesic Cartrophen (0.1 ml of a 1.5 mg/ml stocksolution; Biopharm Australia). Systolic blood pressure (SBP) wasmeasured fortnightly using non-invasive tail-cuff plethysmographyapparatus (MC4000 Blood Pressure Analysis System, Hatteras InstrumentInc) before drug treatment (week 0), at week 2 of treatment and end oftreatment (week 4). At the end of drug treatment, body weight of micewas recorded. Mice were anaesthetized using Isoflurane inhalation andkilled by cervical dislocation. Organs (heart, aorta, kidneys, brain,blood and tibia) were collected, with heart and aorta being dissectedappropriately as described below. All organs were then snap frozen inliquid nitrogen, and stored at −80° C. if they were not used forvascular reactivity studies conducted on the day mice were killed.

The following procedures were conducted on organs harvested from theabove experimental groups:

Cardiac Fibrosis Analysis

To measure collagen deposition, frozen sections of heart, kidney oraorta (all 5 μm thickness) were air dried for 10 minutes and werebrought through 3 times xylene (2 minutes each), and 3 times absolutealcohol washes before being rinsed in tap H₂0 for 30 seconds. Stainingwith an optimal concentration of picrosirius red (in this instance 0.05%picrosirius red diluted in saturated picric acid) was performed and leftfor an hour. Sections were then rinsed in water and differentiated in0.01M HCl for 2 minutes, followed by dehydration via 3 times absolutealcohol washes. Then, slides were brought through 3 times xylene washesbefore being cover slipped according to standard histological techniquesusing DPX as the mounting medium. Images were taken under x20magnification, using bright field (Olympus, BX51) and circularizedpolarized light microscopy (DM IRB, Leica) while percentage of positiveinterstitial collagen staining per total field of view was quantifiedusing ImageJ 1.46 software (Java, NIH), and averaged out from a total ofeight views as the final percentage collagen content in a particularanimal.

Gross Cardiac Hypertrophy Analysis Ventricular weight (VW) was comparedto the body weight (BW) as a ratio of VW:BW (mg/g), as well ascomparison of VW to tibial length (TL) in as a ratio of VW:TL (mg/mm)respectively. The hearts that were embedded in OCT and frozen weretransversely sectioned in a cryostat at 5 μm thickness, and stained withHematoxylin and Eosin (Amber Scientific) for morphological examinationof cell structure. The average of 100 cardiomyocytes per heart sectionwas performed under 60× magnification and analyzed using Image J.

Immunohistochemical Localization of Fibrotic and Inflammatory Markers

Immunostaining was performed on either 5 μm thick transverse frozenheart sections or 5 μm thick frozen thoracic aortic. These sections wereair dried and fixed in ice-cold acetone for approximately 15 minutesbefore washing with 0.01M PBS buffer (3×10 minutes). Sections were thenincubated with 10% goat serum in 0.01M PBS for 30 minutes to reducenon-specific binding. If the primary antibody is raised in goat, thispre-blocked medium is substituted with 5% BSA in PBS and Triton-X. Next,blocking buffers were removed and the primary antibody to respectivemarkers were applied overnight at room temperature based on thefollowing dilution and origin of the antibodies: IRAP (1:500, in-house),α-SMA (1:500, Abcam), Vimentin (1:500, Santa Cruz), P-IκBα (1:200, CellSignalling), F4/80 macrophage (1:100, Serotec), MCP-1 (1:100, SantaCruz), VWF (1:500, abcam). After 4 series of washes in ice-cold PBS onsecond day, appropriate secondary antibodies were incubated with mainlyAlexa 488 (Invitrogen or Abcam), Alexa 594 (Invitrogen) and FluoresceinFI-5000 (Vector) being used. With primary antibodies raised in mouse,another immunofluorescence technique was performed using the mouse onmouse (M.O.M) kit (Vector) on heart sections based on the followingdilution and origin of the antibodies: TGF-β (1:50, Santa Cruz), ICAM-1(1:100, Santa Cruz). All immunofluorescent sections were viewed underx20 magnification on an Olympus, BX51 microscope and images analyzedusing Image J.

Histochemical Localization of Cardiac and Vascular Superoxide

Dihydroethidium (DHE) was used to localize superoxide in situ. 5 μmheart sections or 10 μm thoracic aortic sections were incubated with 2μM DHE for 45 minutes at 37° C. Adjacent section was pre-incubated withPEG-SOD (1000 U/mL) for 30 minutes prior to the 45 minutes incubationwith DHE to confirm specificity of the fluorescent signal forsuperoxide. Fluorescence of the product 2-hydroxyethidium was imagedusing inverted confocal microscope (Nikon, C₁) under excitation emissionspectrum of 568 nm and 585 nm respectively. Laser settings wereidentical for each image acquired and integrated density of thefluorescence was quantified using ImageJ.

Determination of Tissue Protein Expression by Western Blot Analysis

Total proteins from homogenized ventricles were extracted using 1.5×Laemmli buffer containing 25% Glycerol, 7.5% SDS, 250 mM Tris-HCl at pH6.8, and 0.001 g bromophenol blue. Homogenized samples were sonicatedfollowed by heating at 37° C. for 20 minutes and centrifuged at 13,000rpm for 30 minutes at 4° C. RCDC assay was performed and the proteincontent was quantified using ProteinQuant-Lowry software (SoftMax Pro)at 750 nm. Finally, samples were stored at −20° C. Western blot wasperformed firstly with samples (10 or 25 μg/μl/sample) beingelectrophoresed, transferred, and probed with primary antibody TGF-β (25kDA, 1:2000, Santa Cruz), MMP-2 (72 kDA, 1:2000, Millipore), MMP-8 (65kDA, 1:2000, Santa Cruz), MMP-9 (84 kDA, 1:1000, Chemicon), MMP-13 (54kDA, 1:100, Abcam), ICAM-1 (85-110 kDA, 1:200, Santa Cruz), GAPDH (36kDA, 1:20000, Abcam). The secondary antibodies were HRP-conjugated goatanti-mouse IgG (1:10000, Jackson ImmunoResearch) or anti-rabbit IgG(1:10000, DAKO), followed by development with ECL reagent. Membraneswere exposed to CLxPosure film (Pierce, Rockford, Ill.). Immunoreactivebands were then quantified using chemiDoc XRS imager and Quantity Onesoftware (BioRad). Individual bands were quantified using bandsintensity per area and were then normalized to the intensity per area ofthe housekeeping gene GAPDH.

Quantification of Cytokine Expression Profile by Bioplex MultiplexSystem

The levels of cytokines in the heart ventricles and apex were detectedby using the Bio-Plex multiplex assay (Bio-rad). Tissues weresnap-frozen and homogenized with a Bio-Plex cell lysis kit (Biorad)according to the manufacturer's instructions. Briefly, tissues werewashed once with 300 μl of wash buffer and homogenized in lysingsolutions using Tissue Lyser (Qiagen). Samples were left on ice for 30min and centrifuged at 6,000×g for 20 min at 4° C. Supernatant wascollected and protein content was determined using Biorad protein assay(Biorad). 500 μg/ml of protein were used to detect the levels ofcytokines. A panel of Bio-Plex Pro™ Mouse Cytokine Standard 23-Plex,Group I (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10,IL-12(p40), IL-12(p70), IL-13, IL-17A, Eotaxin, G-CSF, GM-CSF, IFN-γ,KC, MCP-1, MIP-la, MIP-113, RANTES, TNFα) was used, containing 23different antibodies covalently coupled to the beads. 50 μl of sample(500 μg/ml) or known standard (200-900 μg/ml) was added to wells of a96-well plate which was pre-coated with the diluted coupled beadsspecific for each antibody and incubated at RT with shaking at 300 RPMfor 30 min in the dark. After washing away any unbound substances,biotinylated detection antibodies were added to create a sandwichcomplex and the plate was incubated for 30 min with shaking at 300 RPMin the dark at RT. Following three washes, the final detection complexwas formed with the addition of streptavidin-phycoerythrin conjugate andincubated for 10 min in the dark at RT with shaking at 300 RPM.Following 3 washes with 100 μl wash buffer, beads were resuspended in125 μl of assay buffer. The samples were read using Bio-Plex MAGPIXMultiplex Reader (Bio-Plex Suspension System). Data were calculated bythe Bio-Plex Manager software.

Determination of Gelatinases Activity by Gel Zymography

Homogenized heart apex in 0.25% Triton X-100 dissolved in 10 mM CaCl₂)and centrifuged at 6000 rpm for 30 minutes at 2° C. Pellet undergoesheat extraction in 0.1M CaCl₂) at 60° C. for 4 minutes, followed bychilling in ice and centrifuged at 20000 rpm for 30 minutes at 4° C.Supernatant was sieved using concentrator (company) and stored at −20°C. MMP zymography was performed by firstly with samples (25μg/μl/sample) being electrophoresed. Gels were then washed twice with0.25% Triton X-100 for 15 minutes each, then left for overnightincubation in incubation buffer at 37° C. Gels were stained with 0.1Coomasie blue for an hour followed by destain with 7% acetic acid thenext day. Optical density of bands was then quantified using chemiDocXRS imager and Quantity One software (BioRad).

Determination of Cardiac Function by Langendorff Isolated HeartPreparation

Mice were injected with heparin (500 IU) 20 min before death by cervicaldislocation. The heart was rapidly excised and immersed in ice-coldphysiological saline solution (PSS). Under a dissecting microscope, theheart and aortic arch were cleared of loose tissue, the pulmonary veinperforated to permit free perfusion of the heart and the heart wasmounted on a Langendorff apparatus (ML870B2, ADInstruments, Bella Vista,NSW, Australia) via a 20 gauge needle. The heart was continuouslyperfused with pre-warmed PSS containing (mM): NaCl 118; KCl 4.7;NaHCO₃25; glucose 11; KH₂PO₄ 1.2; MgSO₄ 1.2; CaCl₂) 1.2 mM and gassedwith O₂ 95% and CO₂ 5% (carbogen) at 37° C. Prior to use, the PSS wasfiltered through a 0.22 μm cellulose acetate filter (Millipore). Theheart perfusion chamber was surrounded by thermostatically controlledwater jacket system that maintained the temperature at 37° C. A fine,200 μm cannula was present in the PSS line for drug delivery (1:10 drugdilution and with a time lag of 1 min to the heart). A Millar pressurecatheter (Millar instruments Inc.) was introduced into the leftventricle via a puncture at the junction of the left atrium andventricle, and connected to a Power lab system (ADInstruments).Perfusion pressure was maintained at 80 mmHg and the preparation wasleft to equilibrate for 20-30 min. Left ventricular developed pressure(LVDP); end diastolic pressure (EDP), heart rate (HR), left ventricularcontractility (+dP/dt) and left ventricular relaxation (−dP/dt) andcoronary flow were recorded continuously.

Determination of Ischemic-Reperfusion Injury in the Isolated LangendorffHeart Preparation

Ischemia was induced by halting perfusion of the heart for 40 min. Thiswas followed by 60 min of reperfusion. Left ventricular developedpressure (LVDP), end diastolic pressure (EDP) and contractility (±dP/dt)were recorded during the 60 min. The heart was removed from theLangendorff apparatus and stopped in diastole by placing in highpotassium (100 mM) PSS for 3 min. It was then glued to a mounting block(via the atria), supported by agar blocks and 1 mm thick slices were cut(Integraslice 7550 mM (Campden Instruments, UK). The slices were placedin 2,3,5-triphenyltetrahydrozolium (TTZ 10 mg/ml) and incubated at 37°C. for 15 min. The slices were stored in 4% paraformaldehyde inphosphate-buffered saline and photographed within 24 hr. Infarct areawas determined using ImageJ software (Centre for Information Technology,NIH, Bethesda, Mass., USA). Infarct area was calculated as:

Infarct area (%)=(total infarct area×100)/(total slice area−luminalarea).

Determination of Cardiac Function by Echocardiography

Echocardiography was performed on young (3 month old) and aged (˜22month old) WT and aged (˜22 month old) global IRAP deficient mice underlight sedation (1% isoflurane in oxygen). Echocardiography was performedusing a 18 to 38 MHz linear-array transducer with a digital ultrasoundsystem (Vevo 2100 Imaging System, VisualSonics, Toronto, Canada).Standard parasternal long- and short-axis views were obtained duringeach echocardiographic examination with conventional echocardiographicmeasurements performed offline by a blinded observer. VisualSonics,Toronto, Canada).

Human Cardiac Fibroblast Cell Culture Studies

Commercially available human cardiac fibroblasts (HCF, Catalog #6300,Sciencell, CA, USA) were grown in T75 flask maintained in an incubatorat 37° C., 5% CO₂. Complete media composition: M199 media (#11150-059,life technologies)+10% FBS (#10437-028, life technologies)+1% FibroblastGrowth Supplement-2 (#2382, ScienCell)+1% penicillin/streptomycin 10,000U/ml antibiotics (#15140-122, Life Technologies). Fresh complete mediawas replenished every alternate day until culture reached 70% confluencein which media is replenished daily until it reached approximately 90%confluence in order to passage/subculture. To subculture, media wasdiscarded and culture was rinsed with warm PBS. After which, culture wasdetached using warm 0.05% Trypsin+EDTA with gentle swirling of flask tomake sure cells were not adherent to surface of flask. Trypsin was thenneutralized with complete media and suspension was then transferred intoa new falcon tube and centrifuged at 1000 rpm for 5 minutes. Supernatantwas discarded and pellet of cells were resuspended with 5 ml of completemedia, followed by cell counting. For subculturing/passaging, 1 millionHCFs are placed into a T75 flask. For Picrosirius Red (PSR) staining orimmunofluorescence experiments, 100 k cells were loaded per well in a 24well plate lined with round coverslips. For western blot analysisexperiments, 100 k cells were loaded per well in a 12 well plate.Passage 3-6 cells had been used for experiments with the pro-fibroticagent Angiotensin II (Ang II; 10⁻⁸M 10⁻⁷M 10⁻⁶M) added in complete mediaat the time when cells were being passaged and plated. All duration oftreatment was approximately 72 hours. Once treatment is done, media wascollected and cells were treated differently depending on the type ofexperiments as follow:

A) Picrosirius Red (PSR) Staining

Cells were initially grown on coverslips, washed with warm PBS once andfixed in ice-cold methanol overnight at −20° C. The next day, methanolwas discarded and cells were washed once with cold PBS and incubatedwith 0.1% PSR solution for 1 hour at room temperature. After this, thedye was removed and cells were washed 3 times with 0.1% acetic acid,followed by dehydration with 3 changes of 100% ethanol (5 minutes each)and 3 times with xylene (10 minutes each). Coverslips were removed andmounted on slides using DPX mounting medium.

B) Immunofluorescence:

Cells were grown on coverslips, washed with warm PBS once and fixed inice-cold acetone for 5 minutes at −20° C. Once acetone was discarded,cells were rinsed in PBS, 3×10 minutes at room temperature. Cells werethen blocked with 10% goat serum for 30 minutes at room temperature,followed by overnight incubation with primary antibody (1:500 dilution)at 4° C. The next day, primary antibody was removed and cells wererinsed with PBS 3×10 minutes at room temperature. Cells were thenincubated with secondary antibody (1:500 dilution) for 2 hours at roomtemperature. Cells were then again rinsed with PBS 3×10 minutes at roomtemperature. Coverslips were removed from 24 well plate and mounted onslides using Vectashield mounting medium with DAPI, left to dry prior toimaging under confocal microscope.

C) Western Blot Analysis:

i. Protein Extraction:

Once treatment is complete, cells were washed with warm PBS and detachedusing Accutase (A6964, Sigma), with 5 minutes incubation at 37° C. Cellswere then collected and centrifuged at 7000 rpm for 5 minutes at 4° C.During this time, 1×RIPA lysis buffer cocktail was prepared fresh. Aftercentrifugation, supernatant was discarded. Cell pellet was then lysed in20 ul of 1×RIPA lysis buffer cocktail and kept on ice for 30 minutes.After that, the cell lysate was centrifuged at 13200 rpm for 10 min at4° C. to pellet nuclei and any insoluble cell debris. The supernatant(20 ul) was transferred to a new tube and protein concentrations weremeasured using Biorad Lowry protein assay. Protein quantification ofrespective markers were performed via standard western blot analysis.

ii. Western Blotting:

10% gels (15 wells) were made up using TGX Stain-Free FastCastAcrylamide starter kit, 10% (#161-0183, Biorad). Samples were preparedby diluting 3 parts sample with 1 part of 4× sample buffer, ie. add 10ul of extracted protein samples (half of total extracted proteins) into3.3 ul of 4×Laemli sample buffer (#161-0747, Biorad). Keep samples onice at all times up till this step. Boil samples at 95° C. for 5minutes. Load all samples onto the 15 wells gel, along with a proteinladder. Make up 1× Running buffer from 10× buffer (#161-0732, Biorad).Top up tank and run samples at 200V for ˜40 mins-1 hour. Terminate gelelectrophoresis once the desired protein bands have been separatedappropriately. Prepare sandwich stacks and membrane (pre-soak membranein methanol for 10 s), then soak them all in 1× Trans-Blot TurboTransfer buffer (#170-4272). Lay a stack of wetted stack on bottom ofcassette (bottom ion reservoir stack), followed by wetted membrane, thenthe gel and lastly with another wetted transfer stack at the top (topion reservoir stack). Roll the assembled sandwich with blot roller toexpel trapped air bubbles. Close and lock cassette lid and insertcassette in the Transfer-Blot Turbo transfer system and begin transfer.Once transfer is completed, wash membranes briefly in TBS-T (0.1%Tween-20 in 1×TBS). Block membranes in blocking buffer (TBS-T/5% skimmilk; 5 g/100 ml) for at least 1 hour at room temperature on amechanical shaker. Replace and incubate the membrane overnight withprimary antibody at 4° C. Next day, wash membrane 3×15 minutes in TBS-T.Incubate secondary antibody in 5% skim milk for 1 hour at roomtemperature on shaker. Wash 3×15 minutes in TBS-T. Incubate membranewith ECL substrate for 5 minutes. Image the membrane with a digitalimager ChemiDoc MP imaging system. Bands were analyzed using Image Labsoftware. Marker of interest such as α-smooth muscle actin (α-SMA) andcollagen type I were quantified against housekeeping gene GAPDH. Allprotein expressions were assessed as a relative ratio to the controlgroup.

Liver Fibrosis—Experimental Design Animals

Male C57BL/6J wild type (WT) mice aged approximately 4 to 6 monthsweighing 30-40 grams were obtained from Monash Animal ResearchLaboratory. Animals were housed in the Animal House in the Department ofPharmacology, Monash University, in standard cages where they wereinitially maintained on a normal diet. The housing was maintained atroughly 21° C.±5° C. with mice exposed to a 12 hour light/dark cycle,and access to food and water ad libitum. Experimental proceduresundertaken were approved and certified by the School of BiomedicalSciences (SOBS) Animal Ethics Committee of Monash University (2013/118).

Experimental Model

A high-salt diet (5% salt) model is a clinically relevant anddisease-reversal model which can replicate the high salt intake byhumans which is currently a growing problem in the developed countries.High salt intake induces changes in the cardiovascular system andinduces remodelling and fibrosis in the heart and liver.

WT mice were placed on a normal rodent diet (ND; 0.5% NaCl) which actedas control or a high salt diet (HSD; 5% NaCl) for a period of 4 weeks.After 4 weeks mice on the HSD were randomised to receive either Vehicle(DMSO/30% HBC solution) or IRAP inhibitor (HFI419; 0.72 mg/kg/d) withboth vehicle and IRAP inhibitor administered via s.c. osmotic mini-pump.Mice continued to be fed a HSD whilst receiving these treatments. At theend of the 8 week treatment period mice were weighed before being killedby overdose of isoflurane inhalation. The liver was removed andsectioned with half of the liver placed in 10% formalin and the restfrozen in liquid nitrogen before being stored in −80° C. freezer forfuture use.

Assessment of Liver Fibrosis

Formalin fixed, paraffin embedded livers were sectioned at thickness of4 μm and were stained with Masson's trichrome according to standardprocedures for analysis of liver fibrosis. Initially sections weredeparaffinised and rehydrated through 100% alcohol, 95% alcohol and 75%alcohol washes then washed in distilled water. Sections were re-fixed inBouin's solution for 1 hour at 56° C. to improve staining quality thenrinsed in running tap water for 5-10 minutes to remove yellow colour.Following this, sections were stained in Weigert's iron hematoxylinworking solution for 10 minutes. Rinsed in running warm tap water for 10minutes. Washed in distilled water. Stained in Biebrich scarlet-acidfuchsin solution for 10-15 minutes. Washed in distilled water.Differentiated in phosphomolybdic-phosphotungstic acid solution for10-15 minutes or until collagen was no longer red. Sections weretransferred directly (without rinse) to aniline blue solution andstained for 5-10 minutes. Rinsed briefly in distilled water anddifferentiated in 1% acetic acid solution for 2-5 minutes. Washed indistilled water. Dehydrated very quickly through 95% ethyl alcohol,absolute ethyl alcohol and clear in xylene. Mounted with DPX mountingmedium.

Quantification of liver fibrosis was performed using images capturedwith the Aperio scanner (Monash Histology Platform, Monash University),with ×5 magnification. Each liver section had 5 different fields of viewphotographed at this magnification. Percentage of interstitial andperivascular collagen was analysed and quantified using ImageJ 1.48software (Java, NIH), and the percentage from 5 random fields of viewwere averaged for final percentage of collagen for that particularanimal. All analysis of collagen expression was conducted in a blindedfashion.

Statistical Analysis

Results were expressed as mean±standard error of mean (SEM). Allstatistical plots and analysis were performed using the Prism program(GraphPad Software Inc. SanDiego, Calif., USA). All statisticalcomparison (cardiac hypertrophy, collagen deposition, all IHCquantifications and western blot analysis) between aged WT and IRAP KOmice in aged models or comparison between vehicle-treated aged WT andHFI-419 treated aged WT in the reversal model was conducted usingT-test. For all data sets comparing between young and aged WT orIRAP^(−/−) as well as data in the endothelial vasodilator function werecompared using 2-way analysis of variance (ANOVA) followed by post-hocBonferroni corrections as appropriate. In the Langendorff isolated heartperfusion experiment, equality of standard deviations and Gaussiondistribution, using the Kolmogorov/Smirnov method, were tested. One- andtwo-way ANOVA with post hoc Bonferonni testing was performed on basalrecordings of LVDP, EDP, HR, +dP/dt, while the LVDP and EDP postischaemia-reperfusion were assessed using 2-way ANOVA.

Compounds

Number Structure HFI- 419

Com- pound 1

Com- pound 2

HFI-419, compound 1 and Compound 2 were synthesised according toWO2009065169, AU 2015901676 and Andersson et al J. Med. Chem., (2010)53, 8059 respectively. The synthesis of some of the compounds are listedbelow and their inhibitory activity described in PCT/AU2016/050332.

General Information

All reagents and solvents were used as received. Proton nuclear magneticresonance (¹H n.m.r.) spectra were recorded at 300 MHz with a BrukerAdvance DPX-300 or at 400 MHz using a Bruker Ultrashield-Advance III NMRspectrometer. The ¹H n.m.r. spectra refer to solutions in deuteratedsolvents as indicated. The residual solvent peaks have been used as aninternal reference, with each resonance assigned according to thefollowing convention: chemical shift (δ) measured in parts per million(ppm) relative to the residual solvent peak. High Resolution MassSpectrometry analyses were collected on a Bruker Apex II FourierTransform Ion Cyclotron Resonance Mass Spectrometer fitted with anelectrospray ion source (ESI). Low Resolution Mass Spectrometry analyseswere performed using a Micromass Platform II single quadrupole massspectrometer equipped with an atmospheric pressure (ESI/APCI) ionsource.

Liquid Chromatography Mass Spectra (LCMS) were measured on a Shimadzu2020 LCMS system incorporating a photodiode array detector (214 nmunless otherwise stated) coupled directly into an electrosprayionisation source and a single quadrupole mass analyser. StandardRP-HPLC was carried out at room temperature employing a Phenomenex LunaC₈ (100×2.0 mm I.D.) column eluting with a gradient of 0-64% CH₃CN in0.05% aqueous trifluoroacetic acid over 10 min at a flow rate of 0.2ml/min unless stated otherwise. Mass spectra were obtained in positivemode with a scan range of 200-2000 m/z. Analytical HPLC was performed ona Waters 2690 HPLC system incorporating a diode array detector (254 nm),employing a Phenomenex column (Luna C₈(2), 100×4.5 mm ID) eluting with agradient of 16-80% acetonitrile in 0.1% aqueous trifluoroacetic acid,over 10 minutes at a flow rate of 1 ml/min. Analytical thin layerchromatography (t.l.c.) was performed on Merck aluminium sheets coatedin silica gel 60 F254 and visualization accomplished with a UV lamp.Column chromatography was carried out using silica gel 60 (Merck).Purity of compounds (295%) was established by either reverse phase HPLCor 1H n.m.r.

General Method

Piperidine (cat.) was added to a solution of malononitrile (1.1 eq.) andaldehyde (1 eq.) in EtOH (3-5 mL) and stirred at ambient temperature for15 min. Ethyl acetoacetate (1.1 eq.) was added and the mixture stirredat ambient temperature for 4 hrs. The volume of solvent was reduced andthe resulting precipitate was collected and washed with cold EtOH togive the title compound. If required, the compound was recrystallisedfrom hot EtOH or triturated with DCM.

4-(2-Amino-3-cyano-5-(ethoxycarbonyl)-6-methyl-4H-pyran-4-yl)benzoicacid

Following the general method, 4-carboxybenzaldehyde (1.0 g, 6.6 mmol),malononitrile (0.48 g, 7.3 mmol), ethyl acetoacetate (0.95 g, 7.3 mmol),piperidine (8 drops), and ethanol (20 mL), gave the title compound as awhite solid (1.7 g, 78%). 1H NMR (300 MHz, MeOH) δ 7.96 (d, J=7.2 Hz,2H), 7.29 (d, J=7.2 Hz, 2H), 4.46 (s, 1H), 4.02 (q, J=6.9 Hz, 2H), 2.39(s, 3H), 1.08 (t, J=6.7 Hz, 3H). MS (ESI) m/z: 329.4 (M+H)+(65%).

3-(2-Amino-3-cyano-5-(ethoxycarbonyl)-6-methyl-4H-pyran-4-yl)benzoicacid

Following the general method, 3-carboxybenzaldehyde (100 mg, 0.66 mmol),malononitrile (48 mg, 0.73 mmol), ethyl acetoacetate (95 mg, 0.73 mmol),piperidine (drops), and ethanol (3 mL), gave the title compound afterrecystallistation from EtOH as a white solid (41 mg, 19%). 1H NMR (600MHz, MeOD) δ 7.89 (d, J=7.2 Hz, 1H), 7.86 (s, 1H), 7.45-7.41 (m, 2H),4.46 (s, 1H), 4.07-3.98 (m, 2H), 2.39 (s, 3H), 1.09 (t, J=7.1 Hz, 3H).MS (ESI) m/z: 329.4 (M+H)+(80%).

Ethyl4-(4-acetoxy-3-methylphenyl)-6-amino-5-cyano-2-methyl-4H-pyran-3-carboxylate

Following the general method, 4-formyl-2-methylphenyl acetate (100 mg,0.56 mmol), malononitrile (41 mg, 0.67 mmol), ethyl acetoacetate (80 mg,0.67 mmol), piperidine (3 drops), and ethanol (5 mL), gave the titlecompound as a white solid (87 mg, 44%). 1H NMR (300 MHz, CDCl₃) δ7.04-6.99 (m, 2H), 6.93 (d, J=7.9 Hz, 1H), 4.46 (bs, 2H), 4.41 (s, 1H),4.15-3.95 (m, 2H), 2.37 (s, 3H), 2.29 (s, 3H), 2.14 (s, 3H), 1.12 (t,J=7.1 Hz, 3H). MS (ESI) m/z: 357.3 (M+H)+(50%); 713.6 (2M+H)+(100%).

Ethyl4-(4-acetoxy-3,5-dimethylphenyl)-6-amino-5-cyano-2-methyl-4H-pyran-3-carboxylate

Following the general method, 4-formyl-2,6-dimethylphenyl acetate (85mg, 0.44 mmol), malononitrile (32 mg, 0.49 mmol), ethyl acetoacetate (63mg, 0.49 mmol), piperidine (2 drops), and ethanol (3 mL), gave the titlecompound as a white solid (146 mg, 90%). 1H NMR (300 MHz, CDCl₃) δ 6.85(s, 2H), 4.47 (bs, 2H), 4.37 (s, 1H), 4.19-3.94 (m, 2H), 2.37 (s, 3H),2.31 (s, 3H), 2.11 (s, 6H), 1.12 (t, J=7.0 Hz, 3H). MS (ESI) m/z: 371.4(M+H)+(55%); 740.8 (2M+H)+(100%).

Ethyl6-amino-5-cyano-2-methyl-4-(4-(pyridin-2-yl)phenyl)-4H-pyran-3-carboxylate

Following the general method, 4-(2-pyridyl)benzaldehyde (250 mg, 1.36mmol), malononitrile (99 mg, 1.50 mmol), ethyl acetoacetate (195 mg,1.50 mmol), piperidine (3 drops), and ethanol (5 mL), gave the titlecompound as a white solid (410 mg, 83%). 1H NMR (300 MHz, CDCl₃) δ8.71-8.64 (m, 1H), 7.93 (d, J=8.4 Hz, 2H), 7.79-7.67 (m, 2H), 7.31 (d,J=8.4 Hz, 2H), 7.22 (ddd, J=6.6, 4.8, 1.6 Hz, 1H), 4.52 (s, 1H), 4.47(s, 2H), 4.03 (q, J=7.1 Hz, 2H), 2.40 (d, J=0.9 Hz, 3H), 1.11 (t, J=7.1Hz, 3H). MS (ESI) m/z: 362.6 (M+H)+(100%).

Ethyl 6-amino-5-cyano-2-methyl-4-(quinolin-2-yl)-4H-pyran-3-carboxylate

Following the general method, 2-quinoline carboxaldehyde (250 mg, 1.59mmol), malononitrile (115 mg, 1.75 mmol), ethyl acetoacetate (228 mg,1.75 mmol), piperidine (3 drops), and ethanol (5 mL), gave the titlecompound as a white solid after recrystallization (302 mg, 57%). 1H NMR(300 MHz, DMSO) δ 8.32 (d, J=8.5 Hz, 1H), 7.98-7.90 (m, 2H), 7.73 (ddd,J=8.5, 6.9, 1.4 Hz, 1H), 7.56 (ddd, J=8.0, 6.9, 1.2 Hz, 1H), 7.40 (d,J=8.4 Hz, 1H), 6.98 (s, 2H), 4.63 (d, J=1.0 Hz, 1H), 3.89 (qd, J=7.1,2.7 Hz, 2H), 2.38 (d, J=0.8 Hz, 3H), 0.88 (t, J=7.1 Hz, 3H). MS (ESI)m/z: 336.4 (M+H)+(100%).

Ethyl 6-amino-5-cyano-2-methyl-4-(quinolin-3-yl)-4H-pyran-3-carboxylate

Following the general method, 3-quinoline carboxaldehyde (50 mg, 0.32mmol), malononitrile (23 mg, 0.35 mmol), ethyl acetoacetate (45 mg, 0.35mmol), piperidine (1 drop), and ethanol (3 mL), gave the title compoundas a white solid (85 mg, 79%). 1H NMR (300 MHz, CDCl₃) δ 8.80 (s, 1H),8.08 (d, J=8.4 Hz, 1H), 7.96 (s, 1H), 7.80 (d, J=8.2 Hz, 1H), 7.69 (t,J=7.6 Hz, 1H), 7.54 (t, J=7.5 Hz, 1H), 4.67 (s, 1H), 4.62 (bs, 2H), 4.03(q, J=7.0 Hz, 2H), 2.43 (s, 3H), 1.11 (t, J=7.0 Hz, 3H). MS (ESI) m/z:336.4 (M+H)+(100%).

Ethyl 6-amino-5-cyano-2-methyl-4-(quinolin-4-yl)-4H-pyran-3-carboxylate

Following the general method, 4-quinoline carboxaldehyde (250 mg, 1.59mmol), malononitrile (115 mg, 1.75 mmol), ethyl acetoacetate (228 mg,1.75 mmol), piperidine (2 drops), and ethanol (5 mL), gave the titlecompound as a white solid (395 mg, 74%). 1H NMR (300 MHz, CDCl₃) δ 8.86(d, J=4.3 Hz, 1H), 8.31 (d, J=8.5 Hz, 1H), 8.12 (d, J=8.4 Hz, 1H), 7.73(t, J=7.5 Hz, 1H), 7.62 (t, J=7.6 Hz, 1H), 7.20 (d, J=4.4 Hz, 1H), 5.38(s, 1H), 4.60 (bs, 2H), 3.92-3.73 (m, 2H), 2.48 (s, 3H), 0.73 (t, J=7.1Hz, 3H). MS (ESI) m/z: 336.2 (M+H)+(100%).

4-(2-Amino-3-cyano-5-(methoxycarbonyl)-6-methyl-4H-pyran-4-yl)benzoicAcid

Piperidine (2 drops) was added to a suspension of4-(2,2-dicyanovinyl)benzoic acid (200 mg, 1.01 mmol) and methylacetoacetate (117 mg, 1.01 mmol) in EtOH (3 mL). The mixture was stirredat ambient temperature for 6 h. The resulting precipitate was collectedand washed with cold EtOH to give a white solid (117 mg). Columnchromatography (SiO₂, EtOAc: MeOH, 9:1) afforded the title compound aswhite solid (78 mg, 25%). 1H NMR (400 MHz, MeOD) δ 7.96 (d, J=8.2 Hz,2H), 7.29 (d, J=8.3 Hz, 2H), 4.46 (s, 1H), 3.57 (s, 3H), 2.39 (s, 3H).LCMS (ESI) m/z: 315.1 (M+H)+(100%).

4-(2-Amino-5-(benzyloxycarbonyl)-3-cyano-6-methyl-4H-pyran-4-yl)benzoicacid

(i) 4-(2,2-dicyanovinyl)benzoic acid

Piperidine (66 μL, 0.67 mmol) was added to a mixture of malononitrile(480 mg, 7.27 mmol) and 4-carboxybenzaldehyde (1.0 g, 6.65 mmol) in EtOH(5 mL). The suspension was heated to reflux for 18 h. After cooling thesolvent was removed in vacuo and taken up in toluene. The resultingprecipitate was collected and washed with toluene and cold EtOH to givethe intermediate as a pale yellow solid (1.28 g, 85%). 1H NMR (400 MHz,MeOD) δ 8.29 (s, 1H), 8.17 (d, J=8.5 Hz, 2H), 8.04 (d, J=8.3 Hz, 2H).

(ii)4-(2-amino-5-(benzyloxycarbonyl)-3-cyano-6-methyl-4H-pyran-4-yl)benzoicacid

Piperidine (5 μL, 0.05 mmol) was added to a suspension of4-(2,2-dicyanovinyl)benzoic acid (100 mg, 0.5 mmol) and benzylacetoacetate (87 μL, 0.5 mmol) in EtOH (3 mL). The mixture was stirredat ambient temperature for 6 h. The resulting precipitate was collectedand washed with cold EtOH to give a white solid (55 mg). Columnchromatography (SiO₂, EtOAc) afforded the title compound as beige solid(31 mg, 16%). 1H NMR (400 MHz, MeOD) δ 7.89 (d, J=8.4 Hz, 2H), 7.28-7.16(m, 5H), 7.02 (dd, J=7.8, 1.7 Hz, 2H), 5.09 (d, J=12.3 Hz, 1H), 4.94 (d,J=12.3 Hz, 1H), 4.45 (d, J=0.9 Hz, 1H), 2.40 (d, J=1.0 Hz, 3H). 13C NMR(100 MHz, MeOD) δ 169.79, 167.03, 160.45, 159.62, 151.19, 137.07,131.11, 130.73, 129.39, 129.22, 129.13, 128.63, 120.59, 108.00, 67.43,58.77, 40.46, 18.71. MS (ESI) m/z: 391.4 (M+H)+(60%).

Benzyl 6-amino-5-cyano-4-(4-cyanophenyl)-2-methyl-4H-pyran-3-carboxylate

(i) 2-(4-cyanobenzylidene)malononitrile

A suspension of malononitrile (111 mg, 1.68 mmol) and4-cyanobenzaldehyde (200 mg, 1.53 mmol) in H2O (10 mL) was stirred at100° C. for 8 h. The resulting precipitate was collected and washed withH2O to give the title compound as a cream solid (228 mg, 83%). 1H NMR(400 MHz, MeOD) δ 8.31 (s, 1H), 8.09 (d, J=8.3 Hz, 2H), 7.93 (d, J=8.5Hz, 2H).

(ii) Benzyl6-amino-5-cyano-4-(4-cyanophenyl)-2-methyl-4H-pyran-3-carboxylate

Piperidine (3 μL, 0.028 mmol) was added to a suspension of theintermediate 2-(4-cyanobenzylidene)malononitrile (50 mg, 0.28 mmol) andbenzyl acetoacetate (48 μL, 0.28 mmol) in EtOH (2 mL). The mixture wasstirred at ambient temperature for 1 h. The resulting precipitate wascollected and washed with cold EtOH to give the title compound as awhite solid (77 mg, 74%). 1H NMR (400 MHz, CDCl₃) δ 7.51 (d, J=8.5 Hz,2H), 7.35-7.26 (m, 3H), 7.21 (d, J=8.3 Hz, 2H), 7.06-7.01 (m, 2H), 5.08(d, J=12.1 Hz, 1H), 4.93 (d, J=12.1 Hz, 1H), 4.54 (s, 2H), 4.49 (d,J=0.8 Hz, 1H), 2.42 (d, J=1.0 Hz, 3H). 13C NMR (100 MHz, CDCl₃) δ165.23, 158.59, 157.62, 148.97, 135.13, 132.65, 128.67, 128.62, 128.46,128.43, 118.86, 118.35, 111.18, 106.64, 66.92, 61.27, 39.06, 18.78.

Benzyl 6-amino-5-cyano-4-(3-cyanophenyl)-2-methyl-4H-pyran-3-carboxylate

(i) 2-(3-cyanobenzylidene)malononitrile

A suspension of malononitrile (111 mg, 1.68 mmol) and 3-formylbenzonitrile (200 mg, 1.53 mmol) in H2O (5 mL) was stirred at 100° C.with microwave heating for 3 min. The resulting precipitate wascollected and washed with H₂O to give the title compound as a whitesolid (225 mg, 82%). 1H NMR (400 MHz, CDCl₃) δ 8.20 (ddd, J=8.0, 1.2,0.6 Hz, 1H), 8.08-8.07 (m, 1H), 7.90 (dt, J=7.8, 1.3 Hz, 1H), 7.79 (s,1H), 7.71 (t, J=7.9 Hz, 1H). MS (ESI) m/z: 178.2 (M−H)− (50%).

(ii) benzyl6-amino-5-cyano-4-(3-cyanophenyl)-2-methyl-4H-pyran-3-carboxylate

Piperidine (3 μL, 0.028 mmol) was added to a suspension of2-(3-cyanobenzylidene)malononitrile (50 mg, 0.28 mmol) and benzylacetoacetate (48 μL, 0.28 mmol) in EtOH (2 mL). The mixture was stirredat ambient temperature for 1 h. The resulting precipitate was collectedand washed with cold EtOH to give the title compound as a white solid(82 mg, 79%). 1H NMR (400 MHz, CDCl₃) δ 7.49 (dt, J=7.1, 1.6 Hz, 1H),7.40-7.27 (m, 6H), 7.10-7.05 (m, 2H), 5.06 (d, J=12.1 Hz, 1H), 4.95 (d,J=12.1 Hz, 1H), 4.59 (bs, 2H), 4.46 (d, J=0.9 Hz, 1H), 2.42 (d, J=1.0Hz, 3H). 13C NMR (100 MHz, CDCl₃) δ 165.23, 158.60, 157.70, 145.39,135.07, 132.36, 131.31, 131.05, 129.55, 128.76, 128.68, 128.49, 118.83,118.39, 112.84, 106.79, 67.04, 61.36, 38.70, 18.83.

4-(3-Acetyl-6-amino-5-cyano-2-methyl-4H-pyran-4-yl)benzoic acid

Piperidine (38 μL, 0.38 mmol) was added to a suspension of4-(2,2-dicyanovinyl)benzoic acid (750 mg, 3.78 mmol) and acetyl acetone(379 mg, 3.78 mmol) in EtOH (5 mL). The mixture was stirred at ambienttemperature for 18 h. The resulting precipitate was collected and washedwith cold EtOH to give the title compound as a white solid (840 mg,75%). ¹H NMR (400 MHz, MeOD) δ 7.99 (d, J=8.4 Hz, 2H), 7.32 (d, J=8.3Hz, 2H), 4.57 (d, J=0.8 Hz, 1H), 2.33 (d, J=0.9 Hz, 1H), 2.10 (s, 2H).MS (ESI) m/z: 297.3 (M −H)⁻ (40%).

A sample of 4-(3-acetyl-6-amino-5-cyano-2-methyl-4H-pyran-4-yl)benzoicacid was dissolved in an aqueous solution of NH₄HCO₃ (2 eq.) andlyophilized to give compound 1. ¹H NMR (400 MHz, D₂O) δ 7.90 (d, J=8.4Hz, 2H), 7.38 (d, J=8.3 Hz, 2H), 4.63 (d, J=0.8 Hz, 1H), 2.32 (d, J=0.9Hz, 3H), 2.20 (s, 3H).3-(3-Acetyl-6-amino-5-cyano-2-methyl-4H-pyran-4-yl)benzoic acid

Piperidine (2 drops) was added to a solution of malononitrile (48 mg,0.73 mmol) and 3-carboxybenzaldehyde (100 mg, 0.66 mmol) in acetonitrile(3 mL) and stirred at ambient temperature for 1 h. Acetyl acetone (75μL, 0.73 mmol) was added and the mixture stirred at ambient temperaturefor 4 h. The volume of solvent was reduced and the resulting residuepurified by column chromatography (SiO₂, CHCl₃: ACN: AcOH, 9:0.7:0.3).The product was obtained as a beige solid (13 mg, 7%). 1H NMR (400 MHz,MeOD) δ 7.92-7.90 (m, 1H), 7.86 (m, 1H), 7.47-7.44 (m, 2H), 4.57 (d,J=0.9 Hz, 1H), 2.33 (d, J=0.9 Hz, 3H), 2.10 (s, 3H).

5-Acetyl-2-amino-6-methyl-4-(quinolin-2-yl)-4H-pyran-3-carbonitrile

(i) 2-(quinolin-2-ylmethylene)malononitrile

A suspension of malononitrile (92 mg, 1.39 mmol) and 2-quinolinecarboxaldehyde (200 mg, 1.27 mmol) in H₂O (5 mL) were stirred at ambienttemperature for 7 h. The precipitate was collected and washed with H₂Oto give the title compound as a green solid (240 mg, 92%). 1H NMR (400MHz, MeOD) δ 8.48 (d, J=8.0 Hz, 1H), 8.37 (s, 1H), 8.16 (d, J=8.6 Hz,1H), 8.00 (d, J=8.2 Hz, 1H), 7.86 (ddd, J=8.5, 6.9, 1.4 Hz, 1H), 7.80(d, J=8.4 Hz, 1H), 7.73 (ddd, J=8.1, 6.9, 1.2 Hz, 1H).

(ii) 5-acetyl-2-amino-6-methyl-4-(quinolin-2-yl)-4H-pyran-3-carbonitrile

Piperidine (2.4 μL, 0.024 mmol) was added to a solution of2-(quinolin-2-ylmethylene)malononitrile (50 mg, 0.24 mmol) and acetylacetone (25 μL, 0.24 mmol) in EtOH (0.5 mL). The mixture was stirred atambient temperature for 6 h. The resulting precipitate was collected andwashed with cold EtOH to give a pale brown solid (24 mg, 33%). 1H NMR(400 MHz, MeOD) δ 8.33 (d, J=8.5 Hz, 1H), 8.03 (d, J=8.5 Hz, 1H), 7.90(dd, J=8.2, 1.2 Hz, 1H), 7.76 (ddd, J=8.5, 6.9, 1.5 Hz, 1H), 7.59 (ddd,J=8.1, 6.9, 1.2 Hz, 1H), 7.44 (d, J=8.5 Hz, 1H), 4.83 (d, J=1.0 Hz, 1H),2.36 (d, J=1.0 Hz, 3H), 2.16 (d, J=3.4 Hz, 3H). MS (ESI) m/z: 306.5(M+H)+(100%).

5-Acetyl-2-amino-4-(3-cyanophenyl)-6-methyl-4H-pyran-3-carbonitrile

Piperidine (3 μL, 0.028 mmol) was added to a suspension of2-(3-cyanobenzylidene)malononitrile (50 mg, 0.28 mmol) and acetylacetone (28 mg, 0.28 mmol) in EtOH (2 mL). The mixture was stirred atambient temperature for 1 h. The resulting precipitate was collected andwashed with cold EtOH to give a white solid (64 mg). Columnchromatography (SiO₂, EtOAc: Hexane, 1:2 followed by 100% EtOH) affordedthe title compound as white solid (36 mg, 46%). 1H NMR (400 MHz, DMSO) δ7.72 (dt, J=7.3, 1.6 Hz, 1H), 7.64 (s, 1H), 7.57 (t, J=7.5 Hz, 1H), 7.53(dt, J=7.9, 1.6 Hz, 1H), 6.99 (bs, 2H), 4.57 (s, 1H), 2.27 (d, J=0.7 Hz,3H), 2.09 (s, 3H). 13C NMR (100 MHz, DMSO) δ 197.93, 158.45, 156.07,146.31, 132.25, 130.92, 130.61, 130.11, 119.53, 118.72, 114.32, 111.62,56.88, 38.29, 30.12, 18.77.

5-Acetyl-2-amino-6-methyl-4-(4-(thiophen-2-yl)phenyl)-4H-pyran-3-carbonitrile

(i) 2-(4-(thiophen-2-yl)benzylidene)malononitrile

Piperidine (2.6 μL, 0.027 mmol) was added to a solution of malononitrile(19 mg, 0.29 mmol) and 4-(2-thienyl)benzaldehyde (50 mg, 0.27 mmol) inEtOH (1.5 mL). The mixture was stirred at ambient temperature for 1 h.The resulting precipitate was collected and washed with cold EtOH togive the intermediate as a yellow solid (53 mg, 83%). 1H NMR (400 MHz,CDCl₃) δ 7.93 (d, J=8.4 Hz, 2H), 7.76 (d, J=8.5 Hz, 2H), 7.72 (s, 1H),7.51 (dd, J=3.7, 1.1 Hz, 1H), 7.44 (dd, J=5.1, 1.1 Hz, 1H), 7.15 (dd,J=5.1, 3.7 Hz, 1H).

(ii)5-acetyl-2-amino-6-methyl-4-(4-(thiophen-2-yl)phenyl)-4H-pyran-3-carbonitrile

Piperidine (2.2 μL, 0.022 mmol) was added to a suspension of theintermediate 2-(4-(thiophen-2-yl)benzylidene)malononitrile (53 mg, 0.22mmol) and acetyl acetone (23 μL, 0.22 mmol) in toluene (1 mL). Themixture was stirred at ambient temperature for 4 h. The resultingprecipitate was collected and washed with toluene to give a pale yellowsolid. Column chromatography (SiO₂, CH₂Cl₂: Et₂O, 95:5) afforded thetitle compound as a white solid (40 mg, 77%). HRMS (ESI+): Found: m/z337.1008 (M+H)+, C₁₉H₁₇N₂O₂S requires m/z 337.1001. 1H NMR (400 MHz,CDCl₃) (7.58 (d, J=8.3 Hz, 2H), 7.30-7.25 (m, 2H), 7.21 (d, J=8.3 Hz,2H), 7.07 (dd, J=5.1, 3.6 Hz, 1H), 4.46 (s, 1H), 4.43 (bs, 2H), 2.32 (d,J=1.0 Hz, 3H), 2.09 (s, 3H).

5-Acetyl-2-amino-6-methyl-4-(quinoxalin-6-yl)-4H-pyran-3-carbonitrile

(i) 2-(quinoxalin-6-ylmethylene)malononitrile

Piperidine (4.7 μL, 0.047 mmol) was added to a solution of malononitrile(34 mg, 0.52 mmol) and quinoxaline-6-carbaldehyde (75 mg, 0.47 mmol) inEtOH (1 mL). The mixture was stirred at ambient temperature for 1 h. Theresulting precipitate was collected and washed with cold EtOH to givethe intermediate as a light brown solid (66 mg, 68%). 1H NMR (400 MHz,CDCl₃) δ 8.97 (s, 2H), 8.55 (d, J=2.1 Hz, 1H), 8.37 (dd, J=8.9, 2.1 Hz,1H), 8.27 (d, J=8.9 Hz, 1H), 8.01 (s, 1H).

(ii)5-acetyl-2-amino-6-methyl-4-(quinoxalin-6-yl)-4H-pyran-3-carbonitrile

Piperidine (1.4 μL, 0.015 mmol) was added to a suspension of theintermediate 2-(quinoxalin-6-ylmethylene)malononitrile (30 mg, 0.145mmol) and acetyl acetone (15 μL, 0.145 mmol) in toluene (1 mL). Themixture was stirred at ambient temperature for 4 h. The resultingprecipitate was collected and washed with cold Et20 to give the titlecompound as a beige solid (38 mg, 86%). HRMS (ESI+): Found: m/z 307.1190(M+H)+, C₁₇H₁₅N₄O₂ requires m/z 307.1195. 1H NMR (400 MHz, CDCl₃) δ8.86-8.81 (m, 2H), 8.11 (d, J=8.7 Hz, 1H), 7.92 (d, J=2.0 Hz, 1H), 7.69(dd, J=8.7, 2.1 Hz, 1H), 4.72 (s, 1H), 4.59 (bs, 2H), 2.36 (d, J=0.9 Hz,3H), 2.13 (s, 3H). 13C NMR (101 MHz, CDCl₃) δ 197.92, 157.70, 156.10,145.54, 145.35, 145.26, 143.25, 142.63, 130.77, 129.91, 127.65, 118.58,114.92, 61.83, 39.73, 30.17, 19.11.2-(3-Acetyl-6-amino-5-cyano-2-methyl-4H-pyran-4-yl)benzoic acid

(i) 2-(2,2-dicyanovinyl)benzoic acid

A suspension of malononitrile (48 mg, 0.73 mmol) and2-carboxybenzaldehyde (100 mg, 0.67 mmol) in H₂O (4 mL) was stirred at100° C. with microwave heating for 3 min. The resulting precipitate wascollected and washed with H₂O to give the title compound as a whitesolid (34 mg, 55%). 1H NMR (400 MHz, MeOD) δ 8.87 (s, 1H), 8.19 (dd,J=7.6, 1.2 Hz, 1H), 7.83-7.78 (m, 1H), 7.75 (td, J=7.5, 1.4 Hz, 1H),7.70 (td, J=7.5, 1.3 Hz, 1H).

2-(3-acetyl-6-amino-5-cyano-2-methyl-4H-pyran-4-yl)benzoic Acid

Piperidine (12.5 μL, 0.125 mmol) was added to a suspension of2-(2,2-dicyanovinyl)benzoic acid (50 mg, 0.25 mmol) and acetyl acetone(25 mg, 0.25 mmol) in EtOH (3 mL). The mixture was stirred for 3 d. Thesolvent was removed in vacuo and the residue taken up in EtOAc andstirred for 18 h. The resulting precipitate was collected and washedwith cold EtOAc to give a pale yellow solid (76 mg). Columnchromatography (SiO₂, ACN: CHCl₃, 2:1, followed by EtOAc: MeOH, 95:5)gave a yellow residue (33 mg). 1H NMR (400 MHz, MeOD) δ 7.94 (dd, J=7.9,1.2 Hz, 1H), 7.52 (td, J=7.6, 1.4 Hz, 1H), 7.31 (td, J=7.7, 1.3 Hz, 1H),7.26 (dd, J=7.9, 1.0 Hz, 1H), 6.02 (d, J=1.0 Hz, 1H), 2.29 (d, J=1.0 Hz,3H), 2.05 (s, 3H).

4-(2-Acetamido-5-acetyl-3-cyano-6-methyl-4H-pyran-4-yl)benzoic acid

A solution of 4-(3-acetyl-6-amino-5-cyano-2-methyl-4H-pyran-4-yl)benzoicacid (250 mg, 0.84 mmol) in acetic anhydride (3 mL) was heated to refluxfor 3 h. The mixture was concentrated under a stream of N2 and thenpoured into ice cold H2O. The aqueous solution was extracted with EtOAc(3×20 mL) and the combined organic extract was washed with brine (20mL), dried (MgSO4), filtered and reduced in vacuo to give a yellow oil.The yellow oil was dissolved in EtOH (5 mL) and hydrazine hydrate (1.3eq.) was added. After stirring for 30 min the suspension was reduced invacuo and taken up in H₂O (10 mL) and extracted with EtOAc (3×10 mL).The combined organic extract was dried (MgSO4), filtered and solventremoved in vacuo to give a yellow oil. Column chromatography (SiO₂,EtOAc: MeOH, 95:5 followed by 100% EtOH) afforded the title compound (20mg, 7%). 1H NMR (400 MHz, MeOD) δ 8.02 (d, J=8.4 Hz, 2H), 7.41 (d, J=8.3Hz, 2H), 4.80 (s, 1H), 2.34 (d, J=0.8 Hz, 3H), 2.15 (s, 3H), 2.08 (s,3H). MS (ESI) m/z: 341.4 (M+H)+(100%).

4-(2-Amino-3,5-bis(ethoxycarbonyl)-6-methyl-4H-pyran-4-yl)benzoic acid

(i) (Z)-4-(2-cyano-3-ethoxy-3-oxoprop-1-enyl)benzoic acid

Piperidine (13 μL, 0.13 mmol) was added to a suspension of ethylcyanoacetate (151 mg, 1.33 mmol) and 4-carboxybenzaldehyde (200 mg, 1.33mmol) in EtOH (3 mL). The mixture was heated to reflux for 3 h. Themixture was concentrated in vacuo. Toluene was added and the resultingprecipitate was collected and washed with toluene to give theintermediate as a white solid (278 mg, 85%). 1H NMR (400 MHz, MeOD) δ8.40 (s, 1H), 8.16 (d, J=8.6 Hz, 2H), 8.10 (d, J=8.4 Hz, 2H), 4.39 (q,J=7.1 Hz, 2H), 1.39 (t, J=7.1 Hz, 3H).

(ii) 4-(2-amino-3,5-bis(ethoxycarbonyl)-6-methyl-4H-pyran-4-yl)benzoicacid

Piperidine (20 μL, 0.2 mmol) was added to a suspension of(Z)-4-(2-cyano-3-ethoxy-3-oxoprop-1-enyl)benzoic acid (50 mg, 0.2 mmol)and ethyl acetoacetate (26 mg, 0.2 mmol) in EtOH (3 mL). The mixture wasstirred at ambient temperature for 2 d.

Piperidine (10 μL, 0.1 mmol) was added and solution stirred for afurther 1 d. The mixture was concentrated in vacuo and the residuepurified by column chromatography (SiO₂, EtOAc: Hexane, 2:1) to give ayellow oil. Recystallisation from EtOH gave a white solid (>5 mg). 1HNMR (400 MHz, MeOD) δ 7.88 (d, J=8.4 Hz, 2H), 7.29 (d, J=8.3 Hz, 2H),4.73 (d, J=0.7 Hz, 1H), 4.12-3.98 (m, 4H), 2.37 (d, J=0.8 Hz, 3H), 1.18(t, J=7.1 Hz, 3H), 1.14 (t, J=7.1 Hz, 3H).MS (ESI) m/z: 372.1(M+H)+(100%).

(v)4-(3-Acetyl-6-amino-5-(ethoxycarbonyl)-2-methyl-4H-pyran-4-yl)benzoicacid

Piperidine (30 μL, 0.3 mmol) was added to a suspension of(Z)-4-(2-cyano-3-ethoxy-3-oxoprop-1-enyl)benzoic acid (50 mg, 0.2 mmol)and acetyl acetone (20 mg, 0.2 mmol) in EtOH (3 mL). The mixture wasstirred at ambient temperature for 24 h. Analytical HPLC shows a 1:1ratio of starting material to product however further reaction timeleads to decomposition. Purification by column chromatography (SiO₂,EtOAc) afforded the title compound (2 mg, 3%).1H NMR (400 MHz, MeOD) δ7.90 (d, J=8.4 Hz, 2H), 7.31 (d, J=8.3 Hz, 2H), 4.79 (s, 1H), 4.13-4.02(m, 2H), 2.32 (d, J=0.7 Hz, 3H), 2.18 (s, 3H), 1.20 (t, J=7.1 Hz, 3H).

IRAP Enzymatic Assay

Crude membranes are prepared from HEK 293T cells transfected with fulllength human IRAP or empty vector, then solubilized in buffer consistingof 50 mM Tris-HCl, 1% Triton X-100, pH 7.4 at 4° C. under agitation over5 h. After solubilization, the membranes are pelleted by centrifugationat 23,100 g for 15 min at 4° C., and the supernatant is reserved as thesource of IRAP activity. The enzymatic activities of IRAP are determinedby the hydrolysis of the synthetic substrate Leu-MCA (Sigma-Aldrich,Missouri, USA) monitored by the release of a fluorogenic product, MCA,at excitation and emission wavelengths of 380 and 440 nm, respectively.Assays are performed in 96-well plates; each well contains between0.2-10 μg solubilized membrane protein, a range of concentration ofsubstrate in a final volume of 100 μL 50 mM Tris-HCl buffer (pH 7.4).Non-specific hydrolysis of the substrate is corrected by subtracting theemission from incubations with membranes transfected with empty vector.Reactions proceed at 37° C. for 30 min and IRAP inhibitory activitydetermined in 96-well microtiter plates with absorbance monitored on aWallac Victor 3 spectrophotometer. The kinetic parameters (K_(m) and V)are determined by non-linear fitting of the Michaelis-Menten equation(GraphPad Prism, GraphPad Software Inc., Calif., USA); finalconcentrations of Leu-MCA of 15.6 μM-1 mM. Inhibitor constants (Kj) forthe competitive inhibitors are calculated from the relationship IC₅₀═K;(1+[S]/K_(m)), where IC₅₀ values are determined over a range ofinhibitor concentrations (10⁻⁹ to 10*⁴ M). K_(m) values of IRAP forLeu-MCA are determined from the kinetic studies. Binding affinities ofthe compounds to IRAP were examined by monitoring the inhibition of thehydrolysis of Leu-MCA in the presence of increasing concentrations ofthe compounds (10*⁸ to 10⁻³ M).

In order to see whether the inhibitors such as small molecules orantibodies are selective or specific for IRAP, the inhibitory activitiesof inhibitors for other zinc-dependent metallopeptidases can bedetermined in 96-well microtiter plates with absorbance monitored on aWallac Victor 3 spectrophotometer. Such assays are described inWO2009/065169 and include glucose-6-phosphate dehydrogenase andhexokinase activity, leukotriene A4 hydrolase assay, aminopeptidase Nassay and angiotensin converting enzyme assay.

Collectively, the studies in the Examples below show that removal orinhibition of IRAP activity has dramatic effects on cardiac and vasculartissue fibrosis and have identified IRAP as a novel target in CVD.

Example 1

Studies were performed to examine the IRAP-specific effects in the heartand vasculature of the Angiotensin II-induced mouse model of fibrosis asinitial proof-of-principle studies. In the genetic deletion model, maleyoung adult WT and IRAP KO mice, aged between 4-6 months were treatedwith either Ang II or saline subcutaneously for a period of 4 weeks viaosmotic mini pump. Blood pressure was taken fortnightly. In thepharmacological inhibition model, WT mice were treated co-treatedsubcutaneously with the synthetic IRAP inhibitor, HFI-419, along withAng II-infusion for 4 weeks. The inhibitor was dissolved in Dimethylsulfoxide (DMSO) and 2-hydroxypropyl-β-cyclodextrin (HBC) at a ratio of1:3.

Ang II infusion was used as a conventional model to ‘stress’ thecardiovascular system as this endogenous hormone contributes to thedevelopment and progression of a range of cardiovascular diseasesincluding hypertension, heart failure, renal failure and vascularstiffening which are well known risk factors for all of the previouscardiovascular diseases mentioned herein. An advantage of this model,over a naturally ageing model, is that there is a rapid development oforgan fibrosis such that the biochemical and clinical features alreadynoted herein are manifested at a quicker rate. Such rapid changes,particularly in organ fibrosis and hypertension, facilitate the testingof genotype and pharmacological inhibition over a 4-week period thatalso serves the purpose to confirm the universality of our findings indifferent preclinical models. Thus, the Ang II infusion model leads toexacerbation of organ fibrosis and dysfunction at a faster rate thanseen with ageing, and is a well-recognised model of hypertension withmultiple cardiovascular pathologies.

Effect of IRAP Deficiency or IRAP Inhibitor Treatment on Blood PressureFollowing Angiotensin II-Infusion

IRAP deficiency or chronic IRAP inhibitor treatment with HFI419attenuates Ang II-induced increase in blood pressure (FIG. 1). Dataexpressed as mean±s.e.m; P values determined by two way repeatedmeasures analysis of variance (ANOVA).

IRAP Expression in Aorta and Heart of Angiotensin II-Infused Mice

IRAP expression is increased in aortae (FIG. 2a ) and hearts (FIG. 2b )of Ang II-infused WT mice. This is shown by quantification of IRAPexpression in medial and adventitial regions of 5 μm thick transverseaortic sections from adult (4-6 month old) WT and IRAP^(−/−) micetreated with Ang II±vehicle/HFI-419 (n=5). Further, the data in FIG. 2bwas derived from quantification of IRAP in 5 μm thick transverse heartsections from adult (4-6 month old) WT and IRAP^(−/−) mice treated withAng II±vehicle/HFI-419 (n=5). Quantification of IRAP expressed aspercent positive stained tissue area. Data expressed as mean±s.e.m; Pvalues determined by two way analysis of variance (ANOVA).

Genetic Deletion and Pharmacological Inhibition of IRAP AttenuatesAngiotensin II-Mediated Aortic Fibrosis and Associated Markers.

Representative images and quantification of positive stainedimmunofluorescence in thoracic aortic sections from adult (4-6 monthold) WT and IRAP^(−/−) mice treated with saline or AngII±vehicle/HFI-419 demonstrated decreased TGF-β₁ and α-SMA expression inred with green showing autofluorescence of elastic lamina (FIG. 3).Collagen staining was determined using picrosirius red and then imagedusing polarised microscopy. Data expressed as mean±s.e.m of percentagepositive stained area (n=5-6). *P<0.05; **P<0.01; ***P<0.001,****P<0.0001 determined by one way ANOVA with Bonferroni correction formultiple comparisons. These findings indicate that Ang II-inducedvascular fibrosis and elevated profibrotic markers and that theseincreases were prevented in IRAP^(−/−) mice or by HFI-419 treatment.

Genetic Deletion and Pharmacological Inhibition of IRAP AttenuatesAngiotensin II-Mediated Inflammation in the Aorta.

Representative images and quantification of positive stainedimmunofluorescence in thoracic aortic sections from adult (4-6 monthold) WT and IRAP^(−/−) mice treated with saline or AngII±vehicle/HFI-419 showing reductions in P-IκBα (marker for NFκBactivation), MCP-1, ICAM-1 and VCAM-1 (vascular cell adhesion protein-1)expression in red with green showing autofluorescence of elastic lamina(FIG. 4). Data expressed as mean±s.e.m of percentage positive stainedarea (n=5-6). *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 determined byone way ANOVA with Bonferroni correction for multiple comparisons.

Genetic Deletion and Pharmacological Inhibition of IRAP AttenuatesAngiotensin II-Mediated Cardiac Hypertrophy and Fibrosis.

IRAP deficiency or IRAP inhibition (using HFI-419) prevented AngII-mediated increase in cardiac hypertrophy as assessed usingcardiomyocyte cross-sectional area in Haematoxylin & Eosin (H&E) stainedtransverse heart sections (n=6) as shown in FIG. 5a . IRAP deficiency orinhibition significantly decreased interstitial collagen expressiondetermined via brightfield microscopy of picrosirius red stainedtransverse heart sections (n=6) as shown in FIG. 5b . Data expressed asmean±s.e.m of percentage positive stained area (n=5-6). *P<0.05;**P<0.01; ***P<0.001, ****P<0.0001 determined by one way ANOVA withBonferroni correction for multiple comparisons.

Genetic Deletion and Pharmacological Inhibition of IRAP PreventsAngiotensin II-Induced Increase in Cardiac Fibrogenic Markers.

FIG. 6 shows representative images and quantification of positivestained immunofluorescence in transverse heart sections from adult (4-6month old) WT and IRAP^(−/−) mice treated with saline or AngII±vehicle/HFI-419 showing no change in vimentin staining (marker forfibroblast expression), decreased α-SMA staining (marker formyofibroblast expression) and decreased perivascular expression ofTGF-β₁ (fibrogenic cytokine) as well as decreased protein expression ofTGF-β₁ (analysed via western blot). Data expressed as mean±s.e.m ofpercentage positive stained area for immunofluorescence anddensitometric analysis of western blots expressed as relative ratio tomean of WT control±s.e.m; (n=5-6). *P<0.05; **P<0.01; ***P<0.001,****P<0.0001 determined by one way ANOVA with Bonferroni correction formultiple comparisons.

Genetic Deletion or Pharmacological Inhibition of IRAP PreventsAngiotensin II-Induced Increase in Cardiac Reactive Oxygen Species (ROS)and Inflammatory Markers.

FIG. 7 shows representative images and quantification of positivestained immunofluorescence in transverse heart sections orquantification of protein levels using western blot analysis from adult(4-6 month old) WT and IRAP^(−/−) mice treated with saline or AngII±vehicle/HFI-419 (n=5-6). IRAP deficiency or IRAP inhibition preventedAng II-induced increase in superoxide generation, had no effect onexpression of the NADPH oxidase isoform, NOX-2, decreased P-IκBαexpression (marker for NFκB activation), decreased both ICAM-1expression and protein content as well as decreasing MCP-1 andmacrophage expression. Data expressed as mean±s.e.m of percentagepositive stained area for immunofluorescence and densitometric analysisof western blots expressed as relative ratio to mean of WTcontrol±s.e.m; (n=5-6). *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001determined by one way ANOVA with Bonferroni correction for multiplecomparisons.

Example 2

Following on from the proof-of-principle studies (Example 1) showingthat IRAP deficiency and direct pharmacological inhibition of IRAP wereeffective in preventing Angiotensin II-mediated cardiac and vascularfibrosis and inflammation, this example now underlines the potentialclinical effectiveness of targeting IRAP. This is demonstrated using anaged model of cardiovascular fibrosis in which global IRAP deficientmice are protected against age-induced increases in cardiac fibrosis andinflammation whilst direct IRAP inhibition completely reversesage-induced cardiac remodeling.

Global IRAP Gene Deletion Protects Against Age-Induced Cardiac Fibrosis

In the current study, IRAP immunofluorescence was present in bothinterstitial and perivascular regions of the heart and was doubled inthe hearts of aged WT mice when compared to their young genotypecontrols (FIG. 8a,b ). The veracity of this effect was confirmed by theabsence of staining in hearts obtained from young adult and agedIRAP^(−/−) mice (FIG. 8a ). Moreover, IRAP expression was co-localizedwith α-smooth muscle actin (α-SMA) expression in both interstitial andperivascular regions, suggestive of it being located on VSMC as well asdifferentiated myofibroblasts. Cardiac fibrosis, assessed by collagencontent using picrosirius red staining and quantified using both brightfield and circularized polarized light microscopy, was evaluated inyoung and aged WT mice as well as in young and aged IRAP^(−/−) mice. Asexpected, aging significantly increased cardiac interstitial fibrosis,by ˜75% (FIG. 9a,b ; FIG. 10a,b ), and also increased perivascularfibrosis, in line with known elevations in ECM in aging hearts (FIG.10c,d ). Such findings highlight the importance of using animal modelsthat follow a natural evolution of CVD. In contrast to the increase incollagen seen in hearts from our aged WT mice, aged IRAP^(−/−) miceexhibited ECM deposition similar to that seen in young adult WT mice(FIG. 9a,b ; FIG. 10a-d ) indicative of an antifibrotic effect in theabsence of IRAP, which was confirmed by a decrease in mature form ofcollagen αl Type I protein level (FIG. 11).

The fibrogenic cytokine TGF-31 is well known to promote thedifferentiation of fibroblast to a more synthetic type of myofibroblast.In this context, IRAP^(−/−) mice exhibited significantly lower TGF-β1protein in the heart, by Western blot (FIG. 11), and more strikingly,4-fold less perivascular expression of TGF-β1, by immunofluorescence, ascompared to aged WT mice (FIG. 12a,b ). While aging did not affect thedegree of vimentin-positive fibroblast expression between WT and IRAPgenotypes, there was increased myofibroblast expression (αSMA-positive)in hearts from aged WT mice (FIG. 12a,b ). In contrast, hearts from agedIRAP^(−/−) mice did not exhibit this age-dependant myofibroblastupregulation, resulting in myofibroblast expression similar to thatfound in hearts from young WT mice (FIG. 12a,b ). These results suggestthat exaggerated collagen production due to increased syntheticmyofibroblast activity contributed to the increased cardiac fibrosisnoted in aged WT hearts, and that this phenomenon was severely bluntedin hearts from aged IRAP^(−/−) mice. Consistent with this notion, usingdouble labeling IHC, it was revealed that IRAP was co-localized withmyofibroblasts, further implicating a potential role of IRAP in alteringmyofibroblast functional activity.

Homeostasis of ECM is maintained by the balance between collagensynthesis and collagen degradation. In the current study similar proteinlevels or enzymatic activity of gelatinases MMP-2 and MMP-9, and ofcollagenase MMP-8 in aged WT and IRAP^(−/−) mice was demonstrated byWestern blot and zymography, (FIG. 11) whereas there was an 50% increasein MMP-13 protein expression in aged IRAP^(−/−) mice compared toage-matched WT controls (FIG. 11c ). MMP-13 is the main collagenasepresent in the heart thus the increased protein expression indicatesgreater collagen degradation in aged IRAP deficient mice. Collectively,these results indicate that IRAP deficiency is protective againstage-mediated cardiac fibrosis by down-regulating collagen synthesis andup-regulating collagen degradation.

IRAP Deficiency Decreases Superoxide Production and RegulatesInflammation

Dihydroethidium (DHE) staining in the heart revealed ˜40% less cardiacsuperoxide (.O₂ ⁻) production in aged IRAP^(−/−) mice compared to agedWT controls (FIG. 13a ). IRAP^(−/−) mouse hearts also expressed lessphospho-IκBa, indicative of reduced NFκB activation (FIG. 13a ) anddecreased inflammation as demonstrated by reduced monocytechemoattractant protein-1 (MCP-1) expression, markedly reducedintercellular adhesion molecule 1 (ICAM-1) expression, by perivascularimmunohistochemistry and Western blot analysis leading to reducedmacrophage infiltration in the heart (FIG. 13). The pattern of cytokinesreleased from the heart was also examined. There were slight increasesin pro-inflammatory cytokines IL-113, IL-17A and TNF-α in hearts of agedIRAP^(−/−) mice (FIG. 14), however there were more marked increases in anumber of anti-inflammatory cytokines, including IL-2, IL-4, IL-5, IL-10and IL-12p40 (FIG. 14; Table 1) providing evidence for ananti-inflammatory phenotype in aged IRAP^(−/−) mice.

TABLE 1 Cardiac cytokine protein levels. Cardiac cytokine protein levelsin hearts from aged WT, aged IRAP^(−/−), vehicle-treated andHFI-419-treated aged WT mice were quantified using a Bioplex cytokineassay (Bio-rad) kit with cytokine levels expressed as mean ± s.e.m inpg/ml. Cytokines are grouped based on pro-inflammatory,anti-inflammatory, colony-stimulating factor and CC chemokine ligandphenotype. Concentration of cardiac cytokines in IRAP^(−/−) mice areexpressed as a relative ratio to mean concentration of aged WT control;while cytokine levels in HFI-419-treated aged WT hearts are expressed asa relative ratio to mean concentration of vehicle-treated aged WT. Ratioof Ratio of WT IRAP^(−/−) IRAP^(−/−) to WT Vehicle HFI-419 HFI toVehicle Pro-inflammatory IL-1a 6.079 ± 0.41 6.764 ± 0.41 1.11  7.34 ±0.475  7.61 ± 0.56 1.03 IL-1b 52.99 ± 6.28 59.32 ± 5.66 1.12 59.78 ±7.913 69.83 ± 3.75 1.16 IL-6 2.272 ± 0.09 2.548 ± 0.08 1.12* 2.823 ±0.153 2.766 ± 0.15 0.98 IL-12 p70 20.55 ± 1.09 23.82 ± 1.29 1.16 26.46 ±4.32  23.49 ± 1.83 0.88 IL-17A 6.553 ± 0.25 8.126 ± 0.33 1.24** 8.462 ±0.792 8.068 ± 0.29 0.95 TNF-a 185.7 ± 6.49 240.3 ± 5.78 1.18 239.1 ±23.62 219.7 ± 9.76 0.92 Anti-inflammatory IL-2 2.971 ± 0.40 5.082 ± 0.821.71* 5.227 ± 1.128 5.849 ± 0.78 1.12 IL-4 2.422 ± 0.07 2.684 ± 0.861.72* 2.782 ± 0.212 2.676 ± 0.06 1.61** IL-5 3.378 ± 0.16  3.95 ± 0.181.17* 4.052 ± 0.453  3.96 ± 0.14 0.98 IL-9  526.7 ± 22.62  517.1 ± 16.63   0.98 441.9 ± 46.68  557.8 ± 31.03 1.19* IL-10 30.43 ± 1.59 41.29 ±2.16 1.36*** 45.26 ± 6.033 40.13 ± 1.36 1.09 IL-12 p40 3.903 ± 0.194.876 ± 0.21 1.25** 3.742 ± 0.187 4.887 ± 0.28 1.31 IL-13 80.14 ± 2.9882.76 ± 2.24 1.03 93.62 ± 4.677 90.19 ± 2.93 0.96 Colony-stimlatingFactor (CSF) G-CSF  1.66 ± 0.05 1.907 ± 0.06 1.15 1.872 ± 0.258 1.912 ±0.08 1.02 GM-CSF 34.18 ± 1.31 37.58 ± 1.01 1.1** 38.68 ± 3.563 38.18 ±1.37 0.99 M-CSF (IL-3) 1.402 ± 0.11 1.479 ± 0.06 1.05 1.528 ± 0.2661.562 ± 0.11 1.02 CC chemokine ligands (CCL) CXCL-1 (KC) 5.398 ± 0.345.849 ± 0.39 1.08 5.828 ± 0.542 6.859 ± 0.34 1.18 CCL-3 (MIP-1a) 1.861 ±0.18  1.99 ± 0.21 1.07  2.84 ± 0.840 2.493 ± 0.35 0.88 CCL-4 (MIP-1b)75.96 ± 9.22  98.8 ± 6.44 1.3 119.3 ± 10.03  92.74 ± 10.92 0.78 CCL-5(RANTES) 2.493 ± 0.20 2.178 ± 0.10    0.87 2.763 ± 0.248 2.839 ± 0.211.03 CCL-11 (Eotaxin)  85.54 ± 12.36  110.5 ± 11.94 1.29 108.9 ± 27.57140.3 ± 8.18 1.29 *P < 0.05, **P < 0.01, ***P < 0.0001 as determined byt-test; n = 9 in each group.

Pharmacological Inhibition of IRAP Reverses Age-Mediated Cardiac Disease

Given that aged mice lacking IRAP exhibited a cardiac phenotype ofreduced ECM, inflammation and oxidative stress compared to theirage-matched WT controls such that their cardiac phenotype resembled thatof their young adult counterparts, the inventors were interested inwhether or not pharmacological inhibition of IRAP with a small moleculeIRAP inhibitor, at a time of established cardiovascular disease, wouldbe able to reverse cardiac fibrosis. To this end, the synthetic IRAPinhibitor HFI-419 was administered for 4 weeks to 20 month old WT micethat had established cardiac fibrosis. Indeed, HFI-419 significantlydecreased IRAP expression (FIG. 8c ), reversed age-induced collagendeposition to the same level seen in young adult mice (FIGS. 15 and 16)or aged IRAP^(−/−) mice (FIG. 9), and also markedly reduced precursorand mature forms of collagen αl Type I (FIG. 17); all consistent withdownregulation of fibrogenic mediators such as synthetic myofibroblasts(FIG. 18) and TGF-31 expression (FIG. 18) following IRAP inhibition.IRAP inhibition had a slightly different effect on collagen degradationto that seen in IRAP deficient mice with a trend towards increasedprotein expression of the collagenase, MMP-8 whilst there was no changein MMP-2, MMP-9 or MMP-13 protein levels. However, HFI-419 treatmentsignificantly decreased TIMP-1 protein levels, (FIG. 17), thus enablingincreased activity of MMPs to provide an overall increase in collagendegradation with inhibition of IRAP.

IRAP inhibition with HFI-419 also reproduced effects on inflammatorymediators exhibited in IRAP^(−/−) mice, with diminished superoxideproduction, NFκB activation, and reduced ICAM-1, MCP-1 and macrophageexpression in aged WT mice that usually exhibited a heightened state ofinflammation (FIG. 13). Moreover, pro- and anti-inflammatory cytokineswere differentially regulated in HFI-419 treated WT mice (Table 1).Compared to the pro-inflammatory cytokine profile from IRAP^(−/−) mice,direct IRAP inhibition did not increase any of the pro-inflammatorycytokines (FIG. 14 and Table 1) however there were marked increases in anumber of anti-inflammatory cytokines, including IL-4, IL-9 and IL-12p40(Table 1) providing evidence for an anti-inflammatory effect mediated byIRAP inhibition that mirrored the phenotype evident in aged IRAP^(−/−)mice.

Structurally Distinct Classes of IRAP Inhibitors are Equally Effectivein Reversing Age-Induced Cardiac Fibrosis

In addition to HFI419, 2 structurally distinct chemical classes of IRAPinhibitors reverse age-induced collagen expression in the heart as shownin FIG. 19. Class 1 inhibitor is compound 1 and has the structure shownherein, whereas class 2 is compound 2 having the structure shown herein.These data show that 3 different small molecule inhibitor of IRAP havebeen shown to reverse collagen expression, a hallmark of fibrosis, in anage-induced model.

IRAP Inhibition and Cardiac Function

To determine if reduced extracellular matrix deposition translated toimproved cardiac function two protocols have been investigated. In thefirst protocol, hearts were isolated from young WT, aged IRAP−/− mice,and aged WT mice treated with either vehicle or HFI-419 for 4 weeks andwere then subjected to ischemic-reperfusion (IR) injury followed byassessment of cardiac function after IR and analysis of IR-inducedinfarction. At baseline there was no difference in HR, LVDP or LVEDP inaged WT (vehicle and HFI-419 treated) or aged IRAP^(−/−) mice. Therecovery of both LVDP and LV±dP/dt in hearts from vehicle treated agedWT mice were significantly impaired over the time course of ischemia andreperfusion compared to effect of IR injury in hearts from young WT mice(FIG. 20 b,c,d), with these markers of LV function significantly reducedcompared with their pre-ischemic baseline level. IRAP deficiency orchronic IRAP inhibitor treatment did not affect recovery of LVDP in thefirst 10 minutes of reperfusion. However, a significant improvement inlatter stages of reperfusion in LVDP and LV±dP/dt was evident from 20min of reperfusion with no significant difference between recovery ofLVDP in hearts from young WT mice and those from aged IRAP^(−/−) or IRAPinhibitor treated mice (FIG. 20 b,c,d). The ability of IRAP deficiencyor chronic IRAP inhibitor treatment to protect against IR injury wasalso evident when infarct area was measured; with both IRAP deficiencyand IRAP inhibition resulting in ˜50% reduction in infarct area comparedto the aged WT control (FIG. 20a ). In the second protocolechocardiography studies were used to determine whether age-inducedchanges in cardiac function in WT mice were reduced in aged mice thatwere globally deficient in IRAP. Hearts were imaged using a number ofanatomical views and imaging modes via echocardiography with thebaseline heart function metrics of the young and aged WT mice similar tothat reported in previous echocardiography studies on mice of advancedage (Dai et al, Circulation. 2009; 119:2789-2797). However, similar tothe protective effect demonstrated in isolated hearts from IRAP−/− miceafter IR injury, aged IRAP−/− mice exhibit improved cardiac functionwith no age-induced decrease in ejection fraction (FIG. 20e ) and atrend for improved left ventricular contractility (assessed viafractional area change; FAC) (FIG. 20f ) when compared to age-matched WTmice (n=4-5), which correlates with the reduced fibrosis evident in thehearts from aged IRAP−/− mice and validates targeting IRAP.

IRAP Deficiency or Inhibition Did not Alter Systolic Blood Pressure,Cardiac Hypertrophy, Cardiomyocyte Hypertrophy and Medial Hypertrophy

There was minimal difference between aged WT and aged IRAP^(−/−) mice(FIG. 21) or HFI-419 treated aged WT mice (FIG. 16) in terms of systolicblood pressure (SBP).

Cardiac hypertrophy, assessed by ventricular weight to body weight(VW:BW) ratio and ventricular weight to tibial length (VW:TL) ratio, aswell as cardiomyocyte hypertrophy quantified as cross-sectional area ofH&E stained cardiomyocytes, were often increased due to ageing but werenot greatly influenced by IRAP deletion or pharmacological inhibition(FIGS. 21 and 16). Therefore, the striking antifibrotic andanti-inflammatory effects of HFI-419 were independent of changes inblood pressure and heart size.

The inventors have demonstrated for the first time that both IRAPdeficiency and pharmacological inhibition of IRAP protected againstcardiac disease. The strength of the current study was the demonstrationthat not only did gene deletion prevent age-induced cardiac fibrosis,but that pharmacological inhibition of IRAP completely reversedage-induced cardiac fibrosis with this latter effect being of greatclinical significance. Indeed, this beneficial cardiac remodeling wasassociated with decreased collagen synthesis and increased collagendegradation, together with reduced cardiac and vascular inflammation.Furthermore, pharmacological inhibition of IRAP translated intofunctional cardiac and vascular improvement. This study shows thatremoval or blockade of IRAP arrests the progression of fibrosis,highlighting the inhibition of IRAP as a novel therapeutic strategy forCVD, particularly in the aging population.

Senescence is a major risk factor for CVD due to prolonged reactivecardiac remodeling, resulting in irreversible fibrosis. The increasedcardiac stiffness and decreased compliance due to excessive buildup ofcollagen exacerbates cardiac dysfunction which may lead to CHF or impederecovery from MI, or contribute to impaired renal function. Indeed,animal senescence represents a clinically-relevant model withestablished cardiac fibrosis and chronic inflammation. The causes ofsuch age-mediated cardiac fibrosis are multifactorial, with cardiacinjury involving a complex interplay between profibrotic cytokines suchas TGF-β and other inflammatory mediators, which then actsynergistically to aggravate cardiac fibrosis. However, pharmacologicaltreatment to reverse existing ECM and organ dysfunction is currently anunmet clinical need, since successful anti-fibrotic therapy needs tosimultaneously target several key mediators. Therefore, considering theprotective vascular or neuroprotective phenotypes mediated via IRAPinhibition by either Ang IV treatment or genetic ablation of IRAP, theinventors have now delineated the role of IRAP deficiency andpharmacological IRAP inhibition in aged mice, by both prevention andinterventions paradigms.

In this context, our current studies have identified that the enzymeIRAP is upregulated in CVD and that inhibition of IRAP counter-regulatesage-related cardiac fibrosis and dysfunction by a number of mechanisms.Collectively, the results of the current study have identified a noveltherapeutic strategy in the treatment of CVD.

It is well established that aging causes cardiac dysfunction, withchronic inflammation and excessive ECM production, resulting in scarringor cardiac fibrosis. Fibrosis occurs predominantly via the upregulationof the potent pro-fibrotic cytokine TGF-β1 which promotes thedifferentiation of vimentin-expressing fibroblasts to αSMA-expressingmyofibroblasts that leads to increased collagen production. However,aged IRAP^(−/−) mice were protected against age-induced increases ininterstitial collagen deposition seen in WT mice. Mechanistically, thiscould be explained by the fact that aged IRAP^(−/−) mice exhibited a‘young adult’ cardiac phenotype, with significantly less myofibroblastdifferentiation and TGF-β₁ expression compared with hearts from aged WTmice. Furthermore, fibroblast proliferation and fibrosis originates fromperivascular regions and progressively extends into adjacentinterstitial spaces within the heart evidenced in mice by increasedperivascular expression of TGF-β₁ and collagen in the aged WT heart,which was abolished in the aged IRAP^(−/−) mice.

The clinical relevance of IRAP as a therapeutic target was confirmedwhen HFI-419 was given to aged WT mice with established cardiacfibrosis, since this intervention fully reversed cardiac fibrosis byabrogating upstream fibrogenic mechanisms, such as myofibroblastdifferentiation and TGF-β expression, in an identical manner to geneticdeletion. Moreover, IRAP was co-localized with myofibroblasts in bothinterstitial and perivascular region of heart, thus providing theanatomical framework for IRAP to modulate myofibroblast expression andECM synthesis. At the same time, ECM is degraded by proteases such asMMPs. In aged mice, IRAP deletion or pharmacological inhibitionincreased MMP-13 and/or MMP-8 and decreased TIMP-1, suggesting thatcollagen degradation, together with decreased collagen synthesis,contributed to the antifibrotic phenotype of aged hearts in the absenceof IRAP.

Fibrosis is often preceded by inflammation, due to infiltration ofinflammatory cells during the initial phase of injury and the subsequentproduction of multiple cytokines. Aging also elevates ROS, whichexacerbates inflammation. NFκB activation increases chemoattractantssuch as MCP-1 and ICAM-1, promoting inflammatory cell infiltration intothe diseased heart whereby monocytes are differentiated into macrophageswhich also produce superoxide and TGF-β₁ that induce myofibroblastdifferentiation and aggravates cardiac fibrosis. Aged IRAP^(−/−) miceexhibited an anti-inflammatory cardiac phenotype and, remarkably,treatment with HFI-419 reversed existing inflammation in the heart, withsimilarly reduced superoxide, phospho-IκBa, MCP-1, ICAM-1 expression,and reduced macrophage infiltration in both experimental models. Thesefindings were generally consistent with the cardiac cytokine analysiswhich indicated relatively greater increases in a number ofanti-inflammatory cytokines than pro-inflammatory cytokines due to IRAPdeletion. More strikingly, HFI-419 elevated anti-inflammatory cytokinesonly. Thus, given the cross-talk between inflammatory and fibroticpathways, it is likely that the prevailing anti-inflammatory state dueto IRAP deletion or inhibition in aged hearts contributes to thenormalization of cardiac fibrosis in both experimental paradigms.Importantly, the anti-inflammatory effect of HFI-419 and IRAP deletionwas also noted in vascular tissue.

Given the cardiovascular protective effects for IRAP inhibition deducedfrom histo-morphological considerations, the inventors also examined ifthese beneficial effects could be translated into cardiac functionalimprovements. It is well established that the heart muscle can bedamaged in response to ischemia-reperfusion (IR) injury, resulting indecreased LVDP following IR injury, which was evident in our aged WTmice following IR, indicating compromised contractility of the fibroticheart. Hearts from IRAP^(−/−) mice or WT mice chronically treated withHFI-419 for 4 weeks showed significant improvement in post-ischemicrecovery of LVDP. The improved functional effects also correlated wellwith reduction in infarct area following IR injury.

In conclusion, genetic deficiency or pharmacological inhibition of IRAPvirtually abolished cardiac fibrosis, with the important finding thatchronic IRAP inhibitor treatment completely reversed age-induced cardiacfibrosis in 2-year old mice. The mechanisms underlying the cardio-,reno- and vaso-protective effects of IRAP inhibition are likely to bemulti-factorial. These effects include an altered balance of the ECM(decreased production and increased degradation) that favours reducedfibrosis, together with a variety of anti-inflammatory effects; all, orsome, of which may result from changes in IRAP substrate levels and/oraltered IRAP signalling pathways. Collectively, these findings suggestthat IRAP plays a key role in the pathogenesis of cardiovascular diseaseand highlight the potential of pharmacological inhibition of IRAP as anovel therapeutic strategy, particularly for difficult-to-treatend-organ damage that occurs with aging and/or hypertension- orcardiovascular-related injury.

Collectively, these studies provide compelling proof-of-principle thatremoval or inhibition of IRAP activity has dramatic effects on cardiac,renal and vascular tissue fibrosis and have identified IRAP as a noveltarget in CVD.

Example 3

Approximately, 1.7 million Australians and 26 million Americans havechronic kidney disease with reduced kidney function. The finalmanifestation of chronic kidney disease (CKD) is renal fibrosischaracterized by tubulointerstitial fibrosis & glomerulosclerosis.

The studies in this Example show that removal or inhibition of IRAPactivity has dramatic effects on kidney fibrosis and have identifiedIRAP as a novel target in CKD.

Regulation of IRAP Expression and Fibrosis in the Kidney of Aged Mice

Similar to Example 2 above regarding cardiac fibrosis, two specificexperimental paradigms were used. Hence the inventors compared thekidney phenotype between aged WT and global IRAP knockout mice (agedbetween 18-22 months) & young WT mice (aged 4-6 months) to determineprevention of age-related kidney fibrosis development. The inventorsalso compared the treatment of WT aged mice with vehicle or with a smallmolecule inhibitor of IRAP to determine therapeutic treatment ofestablished fibrosis and established the effect of IRAP inhibition onreversal of age-related kidney fibrosis.

IRAP expression is increased in kidneys of aged WT mice compared tolevels expressed in kidneys from young WT mice (FIG. 22a ). IRAPexpression tended to be decreased in kidneys of aged WT mice after 4weeks of treatment with the inhibitor of IRAP (HFI-419). Similar toimmunofluorescence studies in the heart, the specificity of the IRAPantibody was confirmed by the absence of staining in kidneys obtainedfrom aged IRAP^(−/−) mice (FIG. 22a ).

IRAP Deficiency and IRAP Inhibitor Treatment in Age-Induced RenalFibrosis

Kidney interstitial fibrosis, assessed by collagen content usingpicrosirius red staining and quantified using bright field microscopy,was evaluated in young WT, aged WT and IRAP^(−/−) mice as well as inaged WT mice treated with either vehicle or HFI-419 (500 ng/kg/min;s.c.) for 4 weeks. As expected, aging significantly increased kidneyinterstitial fibrosis (FIG. 23a ). In contrast to the increase incollagen seen in kidneys from our aged WT mice, aged IRAP^(−/−) miceexhibited ECM deposition similar to that seen in young adult WT mice(FIG. 23a ) indicative of an antifibrotic effect in the absence of IRAPand consistent with the antifibrotic effect seen in hearts from agedIRAP^(−/−) mice (Example 2). Given that aged WT mice have significantincreases in kidney fibrosis and aged mice lacking IRAP demonstrate akidney phenotype of reduced collagen expression similar to that of theiryoung adult counterparts, the inventors were interested in whether ornot pharmacological inhibition of IRAP with a small molecule IRAPinhibitor, at a time of established cardiovascular/renal disease, wouldbe able to reverse kidney fibrosis. To this end, the synthetic IRAPinhibitor HFI-419 was administered for 4 weeks to ˜20 month old WT micethat had established kidney fibrosis. Indeed, HFI-419 displayed asignificant effect to completely reverse age-induced collagen depositionto a similar level seen in young adult WT and IRAP^(−/−) mice (FIGS. 23a& b).

Increased fibrosis can be due to greater differentiation of fibroblaststo a more synthetic type of myofibroblast. In this context, kidneys fromaged WT mice exhibited significantly more αSMA-positive myofibroblastexpression than kidneys from young WT controls (FIG. 24a ). In contrast,kidneys from aged IRAP^(−/−) mice did not exhibit this age-dependantmyofibroblast upregulation, resulting in myofibroblast expressionsimilar to that found in kidneys from young WT mice (FIG. 24a ). Theseresults suggest that exaggerated collagen production due to increasedsynthetic myofibroblast activity contributed to the increased fibrosisnoted in aged WT kidneys, and that this phenomenon was severely bluntedin kidneys from aged IRAP^(−/−) mice. IRAP inhibition with HFI-419 for 4weeks in aged WT mice demonstrated a trend towards reduced αSMA-positivemyofibroblast expression in kidneys when compared to the age-matchedvehicle-treated control mice (FIG. 24b ).

Example 4

To elucidate mechanisms underlying cardio-protective effect of IRAPinhibition in a clinically relevant human model, a primary cell line ofhuman cardiac fibroblasts was studied. The studies were performed toanswer the following questions: Is IRAP present in these cells and doesa pro-fibrotic stimulator increase IRAP expression? Can IRAP inhibitionreduce myofibroblast expression/collagen production?

Increased IRAP Expression in Human Cardiac Fibroblasts Stimulated withAngiotensin II

Representative images showing primary human cardiac fibroblastsstimulated with increasing concentrations of Ang II induced an increasein expression of IRAP (FIG. 25). There is a clear dose dependentincrease in IRAP expression in the human cardiac fibroblasts.

IRAP Inhibitor Dose-Dependently Decreased α-SMA and Collagen Expressionin Human Cardiac Fibroblasts

Pharmacological IRAP inhibition with a small molecule, HFI-419,dose-dependently decreased myofibroblast expression (α-SMA staining) andcollagen production. Representative images showing increased expressionof α-SMA (red; marker for myofibroblasts) and collagen (green) whenhuman cardiac fibroblasts (HCFs) were stimulated with Ang II (0.1 μM)(FIG. 26a ). Combined Ang II and HFI-419 treatment (0.01 to 1 μM)decreased α-SMA and collagen expression. FIG. 26b is quantitative datafrom western blots confirming dose-dependent decrease in proteinexpression of α-SMA and collagen when HCFs were co-treated with AngII+increasing concentrations of HFI-419 (n=10-12). Data expressed asmean±s.e.m; densitometric analysis of western blots expressed asrelative ratio to mean of control cells±s.e.m; *P<0.05; **P<0.01;***P<0.001 determined by one way ANOVA with Bonferroni correction formultiple comparisons.

Example 5

Effect of IRAP Gene Deletion on Liver Steatosis

Male IRAP knockout mice (IRAO KO: global deletion of the gene forinsulin-regulated aminopeptidase), aged 6 months of age, and theirwildtype counterparts, were fed either a high fat diet (HFD) or a normaldiet (ND). After 4 weeks of dietary manipulation, whole body metabolismwas measured in all groups of mice using the Oxymax Lab AnimalMonitoring System (Columbus Instruments, OH, U.S.A.). As expected, micefed the HFD had a decreased respiratory exchange ratio (ratio betweenthe amount of carbon dioxide produced in metabolism and oxygen used) andincreased heat production when compared to ND fed mice but there was nodifference between genotypes over a 48 hr period.

After 12 weeks of dietary manipulation, mice were killed for tissuecollection. Blood, brain, liver, kidneys, gonadal white adipose tissue(visceral fat), inguinal white adipose tissue (subcutaneous fat), brownadipose tissue (thermogenic fat), intestines, heart and aorta werecollected. Tissue weight was different only in the inguinal whiteadipose tissue, with wildtype mice fed the HFD having a significantlyheavier inguinal white adipose tissue deposit than all other groups.

Liver weights were not different between groups but histologicalexamination of this tissue showed greater levels of steatosis in HFD fedmice compared to ND fed mice and the IRAP KO mice on a HFD displayedreduced steatosis compared to WT mice on a HFD (FIG. 27). This showsthat HFD fed mice displayed non-alcoholic fatty liver disease (NAFLD),or early stage non-alcoholic steatohepatitis (NASH), while inhibition ofIRAP in these mice prevented the excess lipid accumulation in vesicles.

Example 6

Pharmacological Inhibition of IRAP Reverses High Salt Induced Increasein Liver Fibrosis

Salt is well known to be an accelerating factor for the progression ofmetabolic syndrome and is implicated in development of cardiovasculardiseases, most likely due to its pro-oxidant properties. Recent evidenceindicates that a high salt diet (HSD) can exacerbate fat and fibrosisaccumulation in the liver of HFD-fed lectin like oxidized low-densitylipoprotein receptor-1 (LOX-1) transgenic (Tg) and apoE knockout (KO)(TgKO) mice, a model used in studies investigating metabolic syndrome(Uetake et al, Lipids in Health and Disease (2015) 14:6). We weretherefore interested in whether a HSD alone induces significant changesin liver fibrosis and would IRAP inhibitor treatment reverse thesefibrotic changes. Feeding a HSD for 8 weeks to WT (C₅₇Bl/6J) micesignificantly increased fibrosis and number of vacuoles in the liverindicating that this model has all the hallmarks of NASH, includingexacerbated fibrosis. The synthetic IRAP inhibitor HFI-419 wasadministered for 4 weeks to 20 week old WT mice that had already beenfed a HSD for an initial 4 weeks to initiate changes in the liver.Indeed, HFI-419 significantly reversed HSD-induced collagen depositionto the same level seen in mice fed a normal chow diet (FIG. 28) andmarkedly reduced indicators of steatosis in the liver (FIG. 28). Theseanti-fibrotic effects are in line with previous findings showing a clearability for the synthetic IRAP inhibitor, HFI-419 to reverse establishedcardiac fibrosis.

1. A method of treating fibrosis in an individual comprisingadministering an inhibitor of insulin-regulated aminopeptidase (IRAP)thereby treating fibrosis.
 2. A method according to claim 1, wherein theindividual is identified as having fibrosis.
 3. A method according toclaim 1, wherein the method reduces progression of, or reverses, atleast one clinically or biochemically observable characteristic offibrosis, thereby treating fibrosis.
 4. (canceled)
 5. A method accordingto claim 3, wherein the clinically or biochemically observablecharacteristic comprises any one of organ dysfunction, scarring,alteration of normal extracellular matrix balance, increase in collagendeposition, differentiation of fibroblasts to myofibroblasts, reductionin the level of matrix metalloproteinases, increase in the level oftissue Inhibitors of matrix metalloproteinases, increased levels ofeither N-terminal or C-terminal propeptide of type I procollagen (PINPor PICP), decreased levels of C-terminal telopeptide of Type I Collagen(CTP or CITP), increased collagen deposition or impaired cardiacfunction measured by various noninvasive imaging techniques, andimpaired renal function measured by increased proteinurea andalbuminurea, decreased glomerular filtration rate, doubling of plasmacreatinine levels.
 6. A method according to claim 5, wherein collagen isa precursor or mature forms of collagen αl Type
 1. 7. A method accordingto claim 1, wherein the fibrosis is age-induced.
 8. A method accordingto claim 1, wherein the fibrosis is stress-induced or injury-induced. 9.A method according to claim 8, wherein the fibrosis is associated withhypertensive heart disease, hypertensive cardiomyopathy or heartfailure, or nephropathy with or without associated diabetes, or otherstress-induced or injury-induced cardiovascular sequelae that mayinvolve a fibrotic response, with or without underlying cardiovasculardisease.
 10. (canceled)
 11. A method according to claim 1, wherein thefibrosis is selected from the group consisting of cardiac fibrosis,liver fibrosis, kidney fibrosis, vascular fibrosis, lung fibrosis anddermal fibrosis. 12-14. (canceled)
 15. A method according to claim 1114,wherein the fibrosis is non-alcoholic steatohepatitis (NASH). 16.(canceled)
 17. A method according to claim 1, wherein the inhibitor ofIRAP directly inhibits the enzymatic activity of IRAP. 18-25. (canceled)26. A compound having a structure according to Formula (I):

wherein A is aryl, heteroaryl carbocyclyl or heterocyclyl, each of whichmay be optionally substituted, when R¹ is NHCOR₈; or quinolinyl,isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridyl,phthalazinyl or pteridinyl, each of which may be optionally substituted,when R¹ is NR₇R₈, NHCOR₈, N(COR₈)₂, N(COR₇)(COR₈), N═CHOR₈ or N═CHR₈; Xis O, NR′ or S, wherein R′ is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted acyl, optionallysubstituted heteroaryl, optionally substituted carbocyclyl or optionallysubstituted heterocyclyl; R₇ and R₈ are independently selected fromhydrogen, optionally substituted alkyl, optionally substituted aryl, orR₇ and R₈, together with the nitrogen atom to which they are attachedform a 3-8-membered ring which may be optionally substituted; R² is CN,CO₀₂R⁹, C(O)O(O)R⁹, C(O)R⁹ or C(O)NR⁹R¹⁰ wherein R⁹ and R¹⁰ areindependently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl,carbocyclyl, heterocyclyl, each of which may be optionally substituted,and hydrogen; or R⁹ and Ro¹ together with the nitrogen atom to whichthey are attached, form a 3-8-membered ring which may be optionallysubstituted; R₃-R₆ are independently selected from hydrogen, halo,nitro, cyano alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl,carbocyclyl, hydroxy, alkoxy, alkenyloxy, alkynyloxy, alkynyloxy,aryloxy, heteroaryloxy, heterocyclyloxy, amino, acyl, acyloxy, carboxy,carboxyester, methylenedioxy, amido, thio, alkylthio, alkenylthio,alkynylthio, arylthio, heteroarylthio, heterocyclylthio,carbocyclylthio, acylthio and azido, each of which may be optionallysubstituted where appropriate, or any two adjacent R³-R⁶, together withthe atoms to which they are attached, form a 3-8-membered ring which maybe optionally substituted; and Y is hydrogen or C₁₋₁₀alkyl, or apharmaceutically acceptable salt or solvate thereof. 27-31. (canceled)32. A compound according to claim 26, wherein the inhibitor compound hasthe structure:

33-43. (canceled)